Formation of array of membranes and apparatus therefor

ABSTRACT

An array of membranes comprising amphipathic molecules is formed using an apparatus comprising a support defining an array of compartments. Volumes comprising polar medium are provided within respective compartments and a layer comprising apolar medium is provided extending across the openings with the volumes. Polar medium is flowed across the support to displace apolar medium and form a layer in contact with the volumes, forming membranes comprising amphipathic molecules at the interfaces. In one construction of the apparatus, the support that comprises partitions which comprise inner portions and outer portions. The inner portions define inner recesses without gaps therebetween that are capable of constraining the volumes comprising polar medium contained in neighbouring inner recesses from contacting each other. The outer portions extend outwardly from the inner portions and have gaps allowing the flow of an apolar medium across the substrate.

In some aspects, the present invention relates to the formation of anarray of membranes comprising amphipathic molecules using an array ofvolumes of polar medium. A further aspect relates to an apparatussuitable for forming an array of membranes. In other aspects, thepresent invention relates to the formation of an array of volumes ofpolar medium. Such an array of volumes of a polar medium may be used ina range of applications, including the formation of membranes comprisingamphipathic molecules.

Spatially defined arrays of small volumes of fluid in the nanolitre topicolitre range may be used in a wide range of biological,pharmaceutical and other analytical applications. A droplet arrayprovides the opportunity to facilitate high throughput processing ofsmall volumes of individual droplets or groups of droplets and may beused for example to compartmentalise reactions, cell sorting andscreening applications such as protein crystallisation, analysis ofblood or spinal fluid and waste processing. The ability to address andreplace the volumes of fluid in the array is an important aspect, forexample for carrying out reactions on the volumes and replenishing thearray. Microfluidic static droplet arrays are disclosed in Lab Chip,2011, 11, 3949.

Lipid bilayers are thin polar membranes formed from two layers of lipidmolecules. Lipid bilayers are found in cell membranes of most livingorganisms and are usually composed of phospholipids. They areimpermeable to most hydrophilic molecules and ions, and enable cells toregulate their salt concentrations and pH by pumping ions across thelipid bilayer using transmembrane proteins known as ion pumps. Lipidbilayers, or more generally bilayers of amphipathic molecules, alsoserve as excellent platforms for a range of experimental studies. Holdenet al, J. Am. Chem. Soc. 2007, 129, 8650-8655 disclose the formation offunctional bionetworks of aqueous droplets comprising lipid bilayersprovided between droplets. Such networks can act as light sensors,batteries and electrical components by incorporating pumps, channels andpores into the bilayers. Sackmann, Science, New Series, Vol 271, No.5245 (Jan. 5, 1996), pp. 43-48 provides a review of the scientific andpractical applications of supported lipid-protein bilayers includingtheir use in electrooptical biosensors. Jung et al, J. Am. Chem. Soc.,2009, 131 (3), 1006-1014 have developed optical assays for the detectionof protein ligand binding on supported bilayers.

The ability to form a membrane of amphipathic molecules between twodroplets of aqueous solution in a hydrophobic medium such as oil hasbeen demonstrated in WO-2008/012552. Each droplet comprises a layer ofamphipathic molecules encapsulating a hydrophilic medium, the dropletbeing provided in a hydrophobic medium. The droplets are brought intocontact to form the membrane of amphipathic molecules therebetween.Electrodes may be provided within the hydrophilic interior of eachdroplet in order to measure ion flow across the bilayer. A droplet arraymay be provided in a container having an array of micromachined dimplesin which individual droplets may rest.

Another application disclosed in WO-2009/024775 is to form membranes ofamphipathic molecules between the volumes of hydrophilic medium in anarray and a layer of hydrophilic medium formed by a hydrated support incontact with the volumes of hydrophilic medium. This document disclosesa method for producing a droplet interface bilayer, wherein droplets areprepared by contacting an oil/lipid solution with an aqueous solutionand the resulting droplets of aqueous solution are brought into contactwith an aqueous agarose gel support layer.

It is desirable to use membranes of amphipathic molecules to holdmembrane proteins. The provision of ion channel nanopores in highlyresistive amphipathic bilayers for the detection of DNA has beenpreviously well documented. Aqueous solutions are provided on eitherside of the amphipathic bilayer and ion flow through the nanopore takesplace under a potential gradient. DNA may be caused to translocate thepore and the change in ion flow during translocation of DNA through thepore may be measured in order to determine its nucleotide sequence. Thelipid bilayer may be suspended across an aperture by methods well knownin the art such as patch clamping or painting. As an alternative,WO-2009/077734 discloses a plurality of individually addressable lipidbilayers formed across an array of microwell apertures, each microwellcontaining an electrode and an aqueous medium in contact with the lipidbilayer.

A first aspect of the present invention is concerned with convenient andeffective formation of an array of membranes comprising amphipathicmolecules.

According to the first aspect of the present invention, there isprovided a method of forming an array of membranes comprisingamphipathic molecules, the method comprising:

providing an apparatus comprising a support defining an array ofcompartments having openings through which polar medium may beintroduced;

disposing polar medium and apolar medium onto the support to providevolumes comprising polar medium within respective compartments so thatthe volumes polar medium are constrained from contacting volumescomprising polar medium in neighbouring compartments, and a layercomprising apolar medium extending across the openings in the support incontact with the volumes comprising polar medium; and

flowing polar medium across the openings in the support to displaceapolar medium and form a layer comprising polar medium extending acrossthe openings in the support in contact with the volumes comprising polarmedium and membranes comprising amphipathic molecules at the interfacesbetween the layer comprising polar medium and the volumes comprisingpolar medium.

Such a method provides a convenient and effective way to form an arrayof membranes comprising amphipathic molecules. Use of an apparatus thatcomprises a support defining an array of compartments having openings,allows an array of volumes comprising polar medium to be disposed withinthe respective compartments through the openings. As a result, thevolumes comprising polar medium are constrained from contacting volumescomprising polar medium in neighbouring compartments, thereby allowingthe volumes of polar medium to be used independently, facilitating arange of array-based applications. Such an apparatus may be made toaccommodate volumes of any selected size. Typically, the volumescomprising polar medium may have an average volume in the range from 0.4pL to 400 nL.

To form membranes comprising amphipathic molecules, there is provided alayer comprising apolar medium extending across the openings in thesupport in contact with the volumes comprising polar medium. Polarmedium is flowed across the openings in the support to displace apolarmedium and form a layer comprising polar medium extending across theopenings in the support in contact with the volumes comprising polarmedium. The membranes comprising amphipathic molecules are formed at theinterfaces between the layer comprising polar medium and the volumescomprising polar medium. In general, and as described further below, theamphipathic molecules may be provided in the layer comprising apolarmedium and/or the polar medium flowed across the openings in thesupport.

This provides a convenient and effective way to form the membranes. Bydisplacing the apolar medium apolar medium by the polar medium, themembranes are reliably formed.

There are now described various methods of forming an array ofmembranes.

Several different methods may be applied for disposing the volumescomprising polar medium within respective compartments. The particularmethod used depends in part on the structure of the support and whetherthe individual volumes of polar medium are preformed prior to additionto the support or formed subsequently following addition of polar mediumto the support. The support may comprise gaps between the compartmentsor alternatively the support may be provided without gaps betweencompartments. A first and second types of possible method will now bedescribed.

In the first type of possible method for disposing the volumescomprising polar medium within respective compartments, the volumes arepre-formed before disposition in the compartments. Some possibletechniques for this are as follows.

In one possible technique, the polar medium and apolar medium may bedisposed onto the support by forming an emulsion of the volumescomprising polar medium in an apolar medium and flowing the emulsionover the support. In this case, volumes comprising polar medium withinthe apolar medium are introduced into the compartments through theopenings. This allows the compartments to be filled in a straightforwardmanner. The dimensions of the individual volumes of polar medium as wellas that of the compartment may be selected such that a single volume ofpolar medium is provided per compartment.

The partitions of the support may comprises gaps that allow flow ofapolar medium between the compartments, as described in more detailbelow. The gaps are chosen to be of a size that constrains the volumesof polar medium within the compartments whereas the apolar medium isable to flow between the gaps.

The emulsion may further comprise the amphipathic molecules. Thisfacilitates the formation of the membranes when polar medium is flowedacross the openings in the support to form the layer comprising polarmedium. The presence of the amphipathic molecules also stabilises theemulsion.

Typically, the emulsion contains more volumes comprising polar mediumthan the number of compartments. The excess of volumes comprising polarmedium assists in filling a reasonably large proportion of thecompartments. Accordingly, to remove the excess volumes comprising polarmedium, the support may be washed with the apolar medium. This washingmay be performed leaving volumes comprising polar medium insidecompartments, and leaving a layer of the apolar medium used for washingas the layer of apolar medium extending across the openings in thesupport in contact with the volumes comprising polar medium.

In another possible technique, volumes comprising polar medium may bedispensed directly into individual compartments, for example by acousticdroplet injection. With this technique, the dispensing may be controlledsuch that the correct number of volumes comprising the polar medium aredispensed without the need to remove excess volumes.

Where the volumes comprising polar medium are preformed, they may bedroplets of an aqueous buffer solution. Such droplets are easy to formand manipulate.

Where the volumes comprising polar medium are preformed, they may bebeads of an aqueous gel. Such beads are again easy to form andmanipulate and may be shaped as desired

Advantageously, the aqueous gel may be a bead, which being relativelyhard, provides advantages in manipulating the volumes comprising polarmedium. Advantages in filling the compartments may be obtained byflowing an emulsion or suspension of the beads over the support underpositive pressure. The use of a bead which resists the pressure permitsrelatively high positive pressures to be used.

In the second type of possible method, the respective volumes comprisingpolar medium may be provided in respective compartments by disposingpolar medium onto the support, so that the polar medium enters into thecompartments through the openings and the layer comprising apolar mediumis provided subsequently, for example by flowing the apolar mediumacross the support, or by another technique such as spraying.

The polar medium may be disposed onto the support by flowing polarmedium across the support. Excess polar medium may thereafter bedisplaced, leaving discrete volumes comprising polar medium in thecompartments. In one example, a gas is flowed across the substrate todisplace the excess polar medium between the step of flowing polarmedium and the step of flowing apolar medium. In another example, theapolar medium is flowed across the substrate layer comprising apolarmedium, this flow itself displacing the excess polar medium.

Alternatively, the polar medium may be disposed onto the support byinjecting discrete volumes comprising polar medium into thecompartments.

An advantage of providing the individual volumes of polar medium in thisway is that the polar medium may be added to the support in the absenceof amphiphilic molecules.

The support may be pre-treated with a pre-treatment apolar medium priorto disposing the respective volumes comprising polar medium in therespective compartments. In the case where the polar medium is disposedonto the support by flowing polar medium across the support,advantageously, the partitions of the support may comprises gaps thatallow flow of apolar medium between the compartments, as described inmore detail below. In this case, a pretreatment may provide some degreeof sealing of the gaps connecting the respective compartments, therebyconstraining the flow of polar medium between the gaps so that the polarmedium enters into the compartments through the openings. This assistsin the eventual formation of discrete volumes of polar medium byreducing the tendency of the volumes in neighbouring compartments tocontact each other. This is particularly beneficial where no amphiphilicmolecules are initially present in the volumes of polar media providedwithin the array of compartments as they may easily converge if theycontact one another.

The addition of pretreatment may also be used change the contact anglebetween the pretreated material of the support and a volume of polarmedium disposed within a compartment. The pretreatment may be used forexample to increase the phobicity of the support to the polar medium andprovide a volume having a more convex shape in order to optimiseformation of the membrane at the interface between the volume of polarmedium and the layer of polar medium. The use of a pretreatment to alterthe phobicity of the support to a desired level permits the use of awider number of materials to be considered in making the support. Thiscan be useful for example in the case where a particular material isdesirable from a manufacturing point of view but does not have thecorrect properties with regards to the polar and apolar media. The layercomprising apolar medium may further comprise the amphipathic molecules.This facilitates the formation of the membranes when polar medium isflowed across the openings in the support to form the layer comprisingpolar medium.

In one example, the apolar medium may comprise the amphipathic moleculesprior to the addition of the layer of apolar medium to the support.Alternatively, the amphipathic molecules may be added to the layer ofapolar medium following addition of the layer to the support.

Where the layer comprising apolar medium further comprises theamphipathic molecules, between the steps of providing a layer comprisingapolar medium extending across the openings in the support in contactwith the volumes comprising polar medium and flowed across the openingsin the support, the apparatus may be left for a period of time in orderto allow the amphipathic molecules to migrate to the interface betweenthe layer comprising apolar medium and the volumes comprising polarmedium.

In another example, the polar medium that is flowed across the openingsin the support may further comprise the amphipathic molecules. Thissimilarly facilitates the formation of the membranes when polar mediumis flowed across the openings in the support to form the layercomprising polar medium.

In yet another example, the pre-treatment of apolar medium may furthercomprise amphipathic molecules, so that the membranes comprisingamphipathic molecules are formed after the step of flowing polar mediumacross the openings in the support to form a layer comprising polarmedium.

The membranes comprising amphipathic molecules may be used for a rangeof applications such as detection of an analyte at the membraneinterface, determination of a property of the membrane interface, orpassage of an analyte across one or more membrane interfaces. In someapplications, the membranes may be used to analyse a sample comprisingan analyte, for example a biological sample.

In one type of application, there may be used membrane proteins, such asion channels or pores that are inserted into the membranes comprisingamphipathic molecules. The membrane proteins may initially be containedin the volumes comprising polar medium or in the layer comprising polarmedium. Alternatively the membrane proteins may be provided in theapolar medium. The membrane proteins typically spontaneously insert inthe membranes comprising amphipathic molecules. Insertion of themembrane proteins into the membrane can be assisted where necessary forexample by means such as the application of a potential differenceacross the membrane.

Some applications may use measurement of electrical properties acrossthe membranes, for example ion current flow. To provide for suchmeasurements, the support may further comprise respective electrodes ineach compartment making electrical contact with the volumes comprisingpolar medium. Other types of measurements may be carried out for exampleoptical measurements such as fluorescence measurements and FETmeasurements. Optical measurements and electrical measurements may becarried out simultaneously (Heron A J et al., J Am Chem Soc. 2009;131(5):1652-3).

In the case that the apparatus comprises respective electrodes in eachcompartment, the pretreatment, where used, preferably does not cover theelectrode surface and is localised elsewhere on the support.

The apparatus may further comprise a common electrode arranged so thatthe common electrode makes electrical contact with the layer comprisingpolar medium, when disposed extending across the support over theopenings

The apparatus may further comprise an electrical circuit connectedbetween the common electrode and the respective electrodes in eachcompartment, the electrical circuit being arranged to take electricalmeasurements. Such electrical measurements may be dependent on a processoccurring at or through the membranes comprising amphipathic molecules.

In the embodiment of forming an array of membranes whereby an emulsionof volumes comprising polar medium in an apolar medium is formed andflowed over the support, a stable emulsion is required in order toprevent the volumes of polar medium from merging with each other.Merging of volumes gives rise to larger volumes which may be unable tobe accommodated in a compartment and which also gives rise to anincreased range of sizes of volumes. Droplet merging may be prevented orminimised by adding amphiphilic molecules to the apolar medium or polarmedium prior to forming the emulsion such that a volume of polar mediumis effectively coated with a layer of amphiphilic molecules. Howeverthis can in some circumstances give rise to an increased electricalresistance between the electrode and the volume of polar medium due theelectrode being coated with amphiphilic molecules. In an alternativeembodiment of forming an array of membranes whereby the individualvolumes of polar medium are provided in the respective compartmentsprior to the addition of amphiphilic molecules, the volumes of polarmedium are able to directly contact the electrode surfaces.

The support may have a variety of advantageous constructions.

The support may comprise a base and partitions extending from the basethat define the compartments and constrain the volumes comprising polarmedium from contacting volumes comprising polar medium in neighbouringcompartments.

In a first possible type of construction of the support, the partitionscomprise inner portions and outer portions, the inner portions defininginner recesses of the compartments without gaps therebetween, thevolumes comprising polar medium being disposed within the inner recessesof the respective compartments, and the outer portions extendingoutwardly from the inner portions defining outer portions of thecompartments and in which gaps allowing the flow of apolar mediumbetween the compartments are formed. Effectively, the the gaps in thepartitions extend partway to the base. This construction has advantagesof providing reliable and controlled formation of the membranes.

Where the apparatus comprises respective electrodes provided in eachcompartment, the electrodes may be provided at the base.

The volumes comprising polar medium may fill the inner recesses. Thegaps between the outer portions assist in filling of the inner recesses.A meniscus may therefore form across the inner recess. Particularadvantage is achieved in the case that polar medium is disposed inrespective compartments by flowing polar medium across the support, andexcess polar medium is displaced by a displacing fluid, which may be agas or may be the apolar medium flowed across the substrate to form thelayer. In this case, the gaps between the outer portions assist inpermitting flow of the displacing fluid across the substrate, so thatthe apolar medium fills the inner recesses.

The gaps between the outer portions assist the formation of membranes byallowing the displacement of apolar medium between the compartments whenthe polar medium is brought into contact with the polar medium in therecesses.

The inner recesses and the outer portions of the partitions may havedimensions selected for the volumes comprising polar medium to form ameniscus across the inner recess and the layer comprising polar mediummay form meniscuses across the outer portions. Those meniscuses extendtowards each other to an extent that brings the layer comprising polarmedium in contact with the volumes comprising polar medium. Thus thegeometry controls the formation of the membranes providing reliabilityin the formation. This also allows control of the size and stability ofthe membranes comprising amphipathic molecules.

The outer portions are set back from the edges of the inner recesses asviewed from the openings. Although not essential, this assists thefunctions described above of assisting in the filling of the innerrecesses by polar medium and in the layer comprising polar mediumforming meniscuses across the outer portions.

The outer portions may be pillars extending from the inner portion.

The support may be designed as follows to facilitate formation of themembranes comprising amphipathic molecules.

Advantageously, the outer ends of the partitions may extend in a commonplane. This improves the adhesion of the layer comprising polar mediumto the support and therefore improves the stability of formation of themembranes comprising amphipathic molecules.

The edges of the outer ends of the partitions provide pinning of thelayer comprising polar medium to the support. Advantageously, to assistsuch pinning, the partitions may be designed so that the total lengthper compartment of the edges of the outer ends of the partitions in thecommon plane is greater than the largest circumference of the largestnotional sphere that can be accommodated within the compartments.

The inner recesses and/or outer portions may have surfaces having apatterning that is arranged to retain apolar medium, for example aplurality of indentations that extend outwardly of the compartments, orin general any microfabricated surface features. The retention of apolarmedium may advantageously be used to change the surface properties ofthe substrate, for example to control the formation of membranes in anapplication where they are formed.

Apolar medium provided retained by the patterning may serve to changethe contact angle between the volumes of polar medium and the support.This can in some embodiments increase the phobicity of the support tothe polar medium and provide volumes of polar medium having a moreconvex outer surface. This may subsequently result in a smaller membraneformed at the interface between the volume of polar medium and the layerof polar medium. The base of the compartments typically do not havemicrofabricated surface features, such that any pretreatment of apolarmedium added to the apparatus is localised and retained at thepartitions. As such contact between the respective electrodes in thebase of each compartment, if present, and the pretreatment, if present,is minimised.

According to a second aspect of the present invention, there is providedan apparatus for forming an array of volumes comprising polar medium,the apparatus comprising a support that comprises partitions whichcomprise inner portions and outer portions, the inner portions defininginner recesses without gaps therebetween that are capable ofconstraining volumes comprising polar medium that may be contained inneighbouring inner recesses from contacting each other, and the outerportions extending outwardly from the inner portions and having gapsallowing the flow of an apolar medium across the substrate.

An apparatus in accordance with the second aspect of the presentinvention may be used as the apparatus in the first aspect of thepresent invention.

In a second possible type of construction of the support, the partitionsmay have gaps extending to the base allowing the flow of apolar mediumbetween the compartments. This facilitates filling of the compartmentswith the volumes comprising polar medium because the gaps allow fordisplacement of the apolar medium that may enter the compartmentsbeforehand

In a third possible type of construction of the support, the partitionsmay have no gaps allowing the flow of apolar medium between thecompartments. This type of construction has the advantage of maximisingthe electrical isolation of the compartments.

According to a third aspect of the present invention, there is providedan apparatus for holding volumes comprising polar medium comprising:

a support comprising a base and partitions that extend from the base anddefine an array of compartments containing an apolar medium; and

at least some of the compartments also containing volumes comprisingpolar medium within the apolar medium that are constrained by thepartitions from contacting volumes comprising polar medium inneighbouring compartments.

The apparatus according to the second and third aspects of the inventionmay be used as a droplet array in a wide range of biological,pharmaceutical or industrial applications, as discussed above.

An apparatus in accordance with the third aspect of the presentinvention may be used as the apparatus in the first aspect of thepresent invention, or in a fourth aspect of the invention, according towhich, there is provided a method of forming an array of volumescomprising polar medium, the method comprising:

providing a support comprising a base and partitions extending from thebase and defining an array of compartments; and

disposing an apolar medium in the compartments and volumes comprisingpolar medium within the apolar medium in at least some of thecompartments so that the volumes comprising polar medium in respectivecompartments are constrained by the partitions from contacting volumescomprising polar medium in neighbouring compartments.

Such a support provides a convenient and effective way to hold an arrayof volumes comprising polar medium within the apolar medium. Thepartitions constrain the volumes comprising polar medium in respectivecompartments from contacting volumes comprising polar medium inneighbouring compartments, thereby allowing volumes, which may beindividual volumes, of polar medium to be used independently,facilitating a range of array-based applications. Such an apparatus maybe made to accommodate volumes of any selected size. Typically, thevolumes comprising polar medium might have an average diameter in therange from 5 μm to 500 μm, or an average volume in the range from 0.4 pLto 400 nL.

The support is easy to fill with the volumes comprising the polarmedium. In one possible technique, the volumes comprising polar mediummay be disposed within the compartments by forming an emulsion of thevolumes comprising polar medium in an apolar medium and flowing theemulsion over the support. This allows the compartments to be filled ina straightforward manner. Typically, the emulsion contains more volumescomprising polar medium than the number of compartments. The excess ofvolumes comprising polar medium assists in filling a reasonably largeproportion of the compartments. Accordingly, to remove the excessvolumes comprising polar medium, the support may be washed with theapolar medium. This washing may be performed leaving volumes comprisingpolar medium inside compartments.

In another technique, volumes comprising polar medium may be dispenseddirectly into individual compartments, for example by acoustic dropletinjection. With this technique, the dispensing may be controlled suchthat the correct number of volumes comprising the polar medium aredispensed without the need to remove excess volumes.

The support may be used to form membranes comprising amphipathicmolecules between the volumes comprising polar medium and a layercomprising polar medium. That is, a layer comprising polar medium may bedisposed extending across the support over the openings of thecompartments and in contact via the amphipathic membrane with at leastsome of the volumes comprising polar medium. The membranes comprisingamphipathic molecules are formed at the interfaces between the layercomprising polar medium and the volumes comprising polar medium.

In an embodiment, the amphipathic molecules may be provided in thevolumes comprising polar medium and/or the apolar medium in order toprovide a layer comprising amphipathic molecules around the volumescomprising polar medium disposed within the compartments prior toprovision of the layer comprising polar medium.

If for example the volumes comprising the polar medium are provided inthe form of liquid droplets in the apolar medium, the presence of alayer of amphipathic molecules around the volumes reduces the tendencyof the volumes to merge with each other. Thus it is preferable that theamphipathic molecules are added to either the apolar medium or thevolumes comprising the polar medium before formation of the droplets inthe apolar medium. If the individual droplets do not contact each otherprior to being introduced into the compartments, the droplets of polarmedium may be provided in the apolar medium in the absence ofamphipathic molecules. In this latter case, the amphipathic moleculesmay be subsequently added, for example in the layer comprising polarmedium, in order to provide a layer of amphipathic molecules around thevolumes comprising the polar medium provided within the apolar medium.

The membranes comprising amphipathic molecules may be used for a rangeof applications such as detection of an analyte at the membraneinterface, determination of a property of the membrane interface, orpassage of an analyte across one or more membrane interfaces In someapplications, the membranes may be used to analyse a sample comprisingan analyte, for example a biological sample.

In one type of application, there may be used membrane proteins, such asion channels or pores that are inserted into the membranes comprisingamphipathic molecules. The membrane proteins may initially be containedin the volumes comprising polar medium or in the layer comprising polarmedium. Alternatively the membrane proteins may be provided in theapolar medium. This causes the membrane proteins to spontaneouslyinsert, after formation of membranes comprising amphipathic molecules.

Some applications may use measurement of electrical properties acrossthe membranes, for example ion current flow. To provide for suchmeasurements, the support may further comprise respective electrodes ineach compartment making electrical contact with the volumes comprisingpolar medium. Other types of measurements may be carried out for exampleoptical measurements such as fluorescence measurements and FETmeasurements. Optical measurements and electrical measurements may becarried out simultaneously (Heron A J et al., J Am Chem Soc. 2009;131(5):1652-3).

A compartment may contain a single volume of polar medium.Alternatively, a compartment may comprise more than one volume of polarmedium, for example two volumes. The volumes comprising polar medium maybe provided one on top of the other. The membranes comprisingamphipathic molecules may be formed at the interfaces between a layercomprising polar medium and the volumes comprising polar medium.Membranes proteins may be provided at the interface between the volumescomprising polar medium to provide an ion or analyte transport pathwaybetween the electrode and the hydrophilic layer.

The compartments of the array may be arranged in various ways, forexample in a square packed, rectangular packed or hexagonal packedarrangement.

The apparatus may further comprise a common electrode arranged so thatthe common electrode makes electrical contact with the layer comprisingpolar medium, when disposed extending across the support over theopenings.

The apparatus may further comprise an electrical circuit connectedbetween the common electrode and the respective electrodes in eachcompartment, the electrical circuit being arranged to take electricalmeasurements. Such electrical measurements may be dependent on a processoccurring at or through the membranes comprising amphipathic molecules.

The support may have a variety of advantageous constructions.

In a first possible type of construction, the partitions may have gapsallowing the flow of apolar medium between the compartments. Thisfacilitates filling of the compartments with the volumes comprisingpolar medium because the gaps allow for displacement of the apolarmedium that may enter the compartments beforehand.

In a construction having gaps, a first possibility is for the gaps toextend to the base. This construction has the advantage that the flow ofapolar medium may occur between the compartments.

In a construction having gaps, a second possibility is for the gaps toextend partway to the base. For example, the support may have aconstruction in which the partitions comprise inner portions definingthe inner portions of the compartments without gaps therebetween andouter portions that extend outwardly from the inner portion defining theinner portions of the compartments and in which said gaps are formed.This construction has the advantage that the electrical isolation of thecompartments is improved whilst still permitting the flow of apolarmedium between the compartments.

In a second possible type of construction, the partitions may have nogaps allowing the flow of apolar medium between the compartments. Thistype of construction has the advantage of maximising the electricalisolation of the compartments.

In this second possible type of construction, the partitions may have aprofile as viewed across the support that comprises, around individualcompartments, one or more salient portions which serve to reduce thecontact between a volume of polar medium and the inner partitionsurface. This reduction in the contact surface area reduces the surfacetension between the volume of polar medium and the inner partitionsurface and enables the volume to move within a compartment more easilyand for example move to the base of the compartment in order to contactthe electrode surface. The dimensions and number of salient portionsprovided around the surface of an inner partition of a compartment mayvary. The reduction in contact between the droplet and the innerpartitions surface enables a larger droplet to be incorporated thanwould have otherwise been possible in the absence of such salientportions.

The partitions may comprise one or more re-entrant portions providingchannels allowing outflow of apolar medium displaced by entry of avolume of polar medium into the compartment. Such a profile isadvantageous in filling of the compartments with the volumes comprisingpolar medium because the re-entrant portions allow for displacement ofthe apolar medium that may enter the compartments beforehand. Thedimensions of a channel may vary. The apolar medium is more easilydisplaced through channels having a greater cross-sectional area. Thepartitions may comprise both one or more salient portions and one ormore re-entrant portions. The dimensions of the one or more re-entrantportions may also determine the extent of surface contact between avolume of the polar medium and the inner partition surface.

The support may be designed as follows to facilitate formation of themembranes comprising amphipathic molecules.

Advantageously, the outer ends of the partitions may extend in a commonplane. This improves the adhesion of the layer comprising polar mediumto the support and therefore improves the stability of formation of themembranes comprising amphipathic molecules.

The edges of the outer ends of the partitions provide pinning of thelayer comprising polar medium to the support. Advantageously, to assistsuch pinning, the partitions may be designed so that the total lengthper compartment of the edges of the outer ends of the partitions in thecommon plane is greater than the largest circumference of the largestnotional sphere that can be accommodated within the compartments.

The dimensions of the openings of the compartments may be selected sothat when the layer comprising polar medium is provided extending acrossthe support over the openings, the layer comprising polar medium forms ameniscus extending into the compartment to an extent that brings thelayer comprising polar medium in contact with at least some of thevolumes comprising polar medium. This allows control of the size andstability of the membranes comprising amphipathic molecules. In theconstruction where the gaps extend partway down the base defining innerand outer portions, the arrangement and dimensions of the outer portionswill determine whether the meniscus is pinned at the outer portions orthe inner portions.

The following comments about the polar and apolar media apply to all theaspects of the present invention.

The polar medium may be a hydrophilic medium. The apolar medium may be ahydrophobic medium. In a particular embodiment, a single volume of polarmedium is provided in a compartment.

The volumes comprising polar medium are typically volumes comprising anaqueous medium, for example an aqueous buffer solution.

The polar medium of respective volumes provided in the compartments maybe the same or different. The volumes may each comprise differentsubstances or differing concentrations of the same substance. Forexample, the volumes comprising polar medium may contain varying amountsof a substance A and the polar layer may comprise a substance B whereina detectable interaction or reaction may occur between A and B. In thisway substance B may pass through the ion-channels into the respectivevolumes comprising the polar medium. By detecting the individualinteractions or reactions, for example a fluorescent signal, a multitudeof ion channel experiments may be carried our simultaneously for exampleto determine an optimal reaction or interaction between B and A.

The layer comprising polar medium may typically comprise an aqueousmedium, for example an aqueous buffer solution.

The polar medium of the layer may be the same or different polar mediumas the respective volumes provided in the compartments. They maycomprise different substances or differing concentrations of the samesubstance.

Embodiments of the present invention will now be described by way ofnon-limitative example with reference to the accompanying drawings, inwhich:

FIG. 1 is an image in plan view of an apparatus holding an array ofvolumes of polar medium;

FIG. 2 is a partial plan view of the support of the apparatus of FIG. 1;

FIG. 3 is a cross-sectional side view of a single compartment of thesupport taken along line in FIG. 2;

FIG. 4 is an isometric projection of an alternative pattern for pillarsof the partitions defining compartments;

FIG. 5 is a plan view of the pillars in the pattern of FIG. 4;

FIGS. 6 and 7 are isometric projections of further alternative patternsfor the pillars;

FIGS. 8 and 9 are plan views of further alternative patterns for thepillars;

FIG. 10 is an isometric projection of an alternative pattern for pillarsof the partitions defining compartments;

FIG. 11 is a plan view of the pillars in the pattern of FIG. 10;

FIG. 12 is an isometric projection of a first alternative constructionfor the partitions;

FIG. 13 is a plan view of the partitions of FIG. 12;

FIG. 14 is a cross-sectional side of a single compartment of a supportof the apparatus in which the partitions have a second alternativeconstruction;

FIG. 15 is an isometric projection of the second alternativeconstruction for the partitions;

FIG. 16 is a plan view of the partitions of FIG. 15;

FIG. 17 is an isometric projection of a modified construction for thepartitions;

FIG. 18 is a plan view of the partitions of FIG. 17;

FIG. 19 is an isometric projection of a modified construction for thepartitions;

FIG. 20 is a plan view of the partitions of FIG. 19;

FIG. 21 is an isometric projection of a modified construction for thepartitions;

FIG. 22 is a plan view of the partitions of FIG. 21;

FIG. 23 is an isometric projection of a modified construction for thepartitions;

FIG. 24 is a plan view of the partitions of FIG. 23;

FIG. 25 is an isometric projection of a modified construction for thepartitions;

FIG. 26 is a plan view of the partitions of FIG. 25;

FIG. 27 is a diagram of a flow cell assembly incorporating an array;

FIG. 28 is an image of droplets of aqueous solution made by amicrofluidic flow junction;

FIG. 29 is a schematic cross-sectional view of an apparatus containing abead protruding out of a compartment;

FIG. 30 is a schematic cross-sectional view of an apparatus containingplural volumes of hydrophilic medium;

FIG. 31 is a cross-sectional view of part of the apparatus provided witha layer of polar medium;

FIG. 32 is a set of schematic side views of a compartment in successivesteps of a method;

FIG. 33 is a set of schematic side views of a compartment in successivesteps of a method;

FIG. 34 is a schematic side view of a compartment having a pre-treatmentapolar medium applied;

FIG. 35 is a side view of a compartment at the start point of a computersimulation;

FIGS. 36(A) and (B) are side views of a compartment during the computersimulation;

FIG. 36C is a confocal image of a compartment in which an inner recessis filled with a volume of polar medium;

FIG. 37 are side views of compartments of different size during acomputer simulation;

FIG. 38 is a set of images of a support in the construction of FIG. 21in which inner recesses are filled with volumes of polar medium;

FIG. 39 are images of a support in the construction of FIG. 19 in whichinner recesses are filled with volumes of polar medium;

FIGS. 40(A) and (B) are images of a support in the construction of FIG.19 after formation of an array of membranes; and

FIG. 40(C) is a schematic side view of a compartment having a formedmembrane.

FIG. 41 is a graph of current against time showing electrical dataobtained for measurement of ion current flow through an MspA nanopore;

FIG. 42 is a diagram of an electrical circuit of the apparatus;

FIG. 43 is a graph of lifetime against size for various volumes of polarmedium;

FIGS. 44( a) to (c) are schematic side views of a droplets of differentsize in a compartment;

FIGS. 45 and 46 are schematic cross-sectional views of the apparatus ofthe type shown in FIGS. 15 and 16;

FIG. 47 is a side view of a meniscus formed across the opening of acompartment;

FIG. 48 shows current traces as a function of time in ms showinghelicase-controlled DNA movement through an MspA-(B2C) nanopore which isinserted in tri-block co-polymer under an applied potential of 180 mV,wherein A and B show examples of two helicase-controlled DNAtranslocations through MspA nanopores.

FIG. 49 shows a current trace showing characteristic block levelscorresponding to the presence (block labeled2) and absence (blocklabeled1) of thrombin;

FIG. 50 shows a Brightfield image of a chip which has been exposed toMspA-(B2C) (SEQ ID NO: 1) nanopores; and

FIG. 51 shows an Brightfield image of chip which has not been exposed toMspA-(B2C) (SEQ ID NO: 1) nanopores.

The specification refers to various sequences as follows.

SEQ ID NO: 1 shows the amino-acid sequence of MspA-(B2C). The amino-acidsequence of MspA-(B2C) is a variant of SEQ ID NO: 2 with the followingmutations G75S/G77S/L88N/Q126R.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of theMS-B 1 mutant of the MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 3 shows one of the polynucleotide sequences used in Example5. It is connected at its 3′ end to the 5′ end of SEQ ID NO: 4 via fourspacer units.

SEQ ID NO: 4 shows one of the polynucleotide sequences used in Example5. It is connected at its 5′ end to the 3′ end of SEQ ID NO: 3 via fourspacer units.

SEQ ID NO: 5 shows the polynucleotide sequence encoding one subunit ofα-hemolysin-E111N/K147N (α-HL-NN; (Stoddart, D. S., et al., (2009),Proceedings of the National Academy of Sciences of the United States ofAmerica 106, p7702-7707).

SEQ ID NO: 6 shows the amino acid sequence of one subunit of α-HL-NN.SEQ ID NO: 7, shown below, is the polynucleotide sequence of an aptamer,where X is an abasic site.XXXXXXXXXXXXXXXXXXXXXXXAAAAAAAGGTTGGTGTGGTTGG. This sequence does notcomply with WIPO ST. 25 and so has not been included in the sequencelisting.

SEQ ID NO: 8 shows the polynucleotide sequence of a strand of DNA. Thestrand has a BHQ1 label attached to the thymine at position 1 in thesequence and a FAM label attached to the thymine at position 15 in thesequence.

SEQ ID NO: 9 shows the polynucleotide sequence encoding the MspA-(B2C)mutant MspA monomer. The amino-acid sequence of MspA-(B2C) is a variantof SEQ ID NO: 2 with the following mutations G75S/G77S/L88N/Q 126R.

SEQ ID NO: 10 shows the polynucleotide sequence encoding the MS-B1mutant of the MspA monomer. This mutant includes the followingmutations: D90N, D91N, D93N, D118R, D134R and E139K.

FIG. 1 shows an apparatus 1 holding an array of volumes 2 of a polarmedium in apolar medium. The apparatus 1 comprises a support 3 providingan array of compartments 4. In use, all the compartments 4 containapolar medium. At least some of the compartments 4 (in this example mostof the compartments 4) contain single volumes 2 of a polar medium in theapolar medium.

The construction of the support 3 is shown in more detail in FIGS. 2 and3. The support 3 comprises a base 5 and partitions 6 that extend fromthe base 5. The partitions 6 comprise plural pillars 7 that extend outfrom the base 5 as shown in FIG. 3, in this example perpendicularly. Thecompartments 4 have openings provided at the distal ends of the pillars7. These openings provide communication from the compartments 4 into thespace adjacent the support 3, and volumes of polar medium may beintroduced into the compartments 4 through the openings.

The pillars 7 may have different shapes as shown in FIG. 2 so that theydefine the compartments 4 in a regular square array. The pillars 7 areshaped so that they constrain the volumes 2 of polar medium in thecompartments 4 from contacting volumes 2 comprising polar medium inneighbouring compartments. In this example, the pillars 7 include across-shaped pillar 7 a in the corners of compartments 4 with armsprotruding into the compartment 4 and further pillars 7 b along the eachside of the compartment 4, with gaps 8 between the cross-shaped pillars7 a and the further pillars 7 b. The compartments 4 are arranged suchthat the volumes 2 of polar medium are physically separated from eachother. This prevents the volumes 2 of polar medium from merging orcontacting each other to form interfaces. This provides a very stablearray of volumes 2 of polar medium which is capable of being stored overa long period of time. Herein, the terms “inner” and “outer” describerelative locations within the compartments 4 from the openings at theouter end towards the base 5 at the inner end.

The pillars 7 have gaps 8 therebetween. In this example, the gaps 8extend the entire distance from the openings to the base 5. The gaps 8are of sufficient size to allow the flow of an apolar medium between thecompartments 4, whilst maintaining the separation of the volumes 2 ofpolar medium in the compartments 4. The provision of gaps 8 allows theapolar medium to flow between the compartments 4. This greatly aids infilling of the compartments 4 as apolar medium may be displaced by avolume 2 of polar medium entering a compartment 4 through an opening.The gaps 8 also allow the level of apolar medium in the support 3 to becontrolled and equalised across the array. The gaps 8 between thepillars 7 are such that the volumes 2 of polar medium are constrainedfrom moving through the gaps 8 between the compartments 4 or fromcontacting volumes 2 comprising polar medium in neighbouringcompartments.

Optionally, a dam 10 may be provided around the perimeter of the support3 which aids in filling the peripheral edges of the support 3 withapolar medium. One or more channels 11 may be provided in the dam 10through which apolar medium may be introduced or drained from thesupport 3.

The support 3 may be prepared from a range of different materials havinga high electrical resistance, including without limitation undopedcrystalline silicon (i.e. a silicon wafer), SU8, polycarbonate, and/orpolyester, and including any combination of these or other materials.The support 3 may be manufactured using conventional techniques for suchmaterials, including, without limitation, deposition and removaltechniques for example etching or laser processing.

As shown in FIG. 3, the base 5 comprises a substrate 12. The substrate12 supports an electrode 13 in each compartment 4. In this example, theelectrodes 13 are shown recessed into the substrate 12, but they couldalternatively be deposited as an outer layer on an exposed surface ofthe substrate 12. The electrodes 13 are provided to make electricalcontact with the volumes 2 of polar medium contained in the compartments4 and are discussed in more detail below.

The substrate 12 may comprise a surface coating 14 that is optional. Thesurface coating 14 may provide a high resistance outer layer. Onepossible combination of materials for the base 5 is that the base ismade of undoped crystalline silicon (i.e. a silicon wafer) and thecoating 14 to be made of SU8. In the example shown in FIG. 2, thesurface coating 14 is provided on top of the substrate 12 and so hasapertures 15 aligned with the electrodes 13 to allow electrical contactbetween the electrodes 13 and the volumes 2 of polar medium. As analternative the electrodes 12 could be patterned in the same layer asthe surface coating 14 or on top of the surface coating 14.

The partitions 6 may be made of the same or different material to thebase 12 of the support 3 and may have the same or different surfaceproperties. The partitions 6 are typically apolar and may be made forexample from Permex. The partitions 6 may optionally comprise a surfacecoating (not shown) to modify their electrical and/or physicalproperties.

The particular shapes and arrangement of the pillars 7 shown in FIG. 2is not essential and the pillars 7 may have a variety of differentshapes to define the compartments 4 so as to constrain the volumes 2 ofpolar medium in the compartments 4 from contacting volumes 2 comprisingpolar medium in neighbouring compartments. By way of example, FIGS. 4 to8 show some examples of alternative shapes and arrangements for thepillars 7, as follows.

FIGS. 4 and 5 show a support 3 wherein the partitions 6 comprise pillars7 including cross-shaped pillars 7 a and further pillars 7 b in asimilar arrangement to FIG. 2. Thus the pillars 7 are combined withshort and long pitches to prevent merging of the volumes 2 of polarmedium and improve pillar stability.

FIGS. 6 to 9 show other supports 3 in which the pillars 7 have modifiedshapes and patterns. In each case, pillars 7 have gaps 8 that extend theentire distance from the openings to the base 5. The pillars 7 arearranged in a pattern that defines compartments 4 in regions of thesupport 3 where the pillars 7 are widely spaced from each other. Thegaps 8 between the pillars 7 are such that the volumes 2 of polar mediumare constrained from moving between the compartments 4 or fromcontacting volumes 2 comprising polar medium in neighbouringcompartments. In FIG. 6, the partitions 6 comprise an array of circularpillars 7 d.

In FIG. 7 the partitions 6 comprise an array of tri-star pillars 7 g.The tri-star pillars 7 g have three arms with curved re-entrant sidesand enlarged ends. The tri-star pillars 7 g define a plurality ofcompartments 4, with three tri-star pillars 7 g equi-spaced around eachcompartment. In FIGS. 8 and 9, the partitions 6 comprise an array ofcross-shaped pillars 7 h and T-shaped pillars 7 i, respectively,defining a plurality of compartments 4. The cross-shaped pillars 7 g andT-shaped pillars 7 h have re-entrant sides.

In these examples of FIGS. 7 to 9, the number of pillars 7 that arerequired to provide a compartment 4 is less than for example thearrangement of FIG. 2 or 6. The provision of a reduced number of pillars7 makes fabrication of the array easier and increases the mechanicalresilience of the individual pillars.

It has also been found that the circular pillars as shown in FIG. 6 aremechanically less resilient and are more prone to collapse, ordistortion than the more structurally resilient pillars of for exampleFIG. 4 or FIGS. 7 to 9, especially for pillars 7 of heights of the orderof 100 μm and pillar widths of the order of 25 μm. Pillars 7 having ahigher width:height ratio are therefore preferred. FIGS. 11 and 12 showa support 3 wherein the partitions 6 comprise pillars 7 includingcross-shaped pillars 7 a and further pillars 7 b having the same overallarrangement as FIG. 4 except for a modification that the surfaces 62 ofthe cross-shaped pillars 7 a and further pillars 7 b are micro-patternedwith a patterning as follows. In particular, those surfaces 62 areindented with a plurality of indentations 63 that extend outwardly ofthe compartment 4, along the entire length of the cross-shaped pillars 7a and further pillars 7 b. In this example, the indentations 63 arerectangular in cross-section.

The surfaces 62 between the indentations 63 lie in a common curved planeextending around the compartment 4. These surfaces 62 physicallyconstrain a volume 2 of polar medium inside the compartment 4. Thus, thedimensions of the surfaces 62 control the size of the volume 2 of polarmedium that may be accommodated in the compartment 4.

The indentations 63 hold apolar medium that reduces the surface area ofthe partitions 6 that is in contact with a volume 2 of polar medium.This modifies the surface properties of the pillars 7, repelling polarmedium and therefore assisting in constraining a volume 2 of polarmedium held in the compartment 4, and in allowing entry of the polarmedium into the compartment. In general, the patterning could compriseother surfaces features to achieve this effect.

The indentations 63 and surfaces 62 have widths that are small comparedto the size of the volume of volume 2 of polar medium held in thecompartment 4. The indentations 63 and surfaces 62 have widthspreferably of at most 20 μm, more preferably of at most 10 μm. Forexample, if the dimensions of a compartment 4 are characterised withreference to the diameter d of the largest notional sphere that can beaccommodated within the compartment 4, then the indentations 63 andsurfaces 62 have widths that are at most 0.1d, preferably at most 0.05d.In a typical example where the diameter d is 140 μm, the indentations 63and surfaces 62 have widths that are 5 μm. The depth of the indentations63 is chosen to allow the channels to retain the apolar medium. In theexample of FIGS. 11 and 12, the channels have a depth of 5 μm, providingan aspect ratio of 1:1. However, deeper indentations 63 provide moreeffective retention of apolar medium.

In all the constructions shown in the figures, the pillars 7 have thesame height so that the outer ends 9 of the pillars 7 extend in a commonplane, as shown in FIG. 3, to provide the support 3 with a brush-likeplanar upper surface. Whilst the provision of pillars having the sameheight is a preferred construction, constructions may be provided havingpillars of differing heights.

There will now be described some alternative constructions for thepartitions 6 in which the partitions 6 do not have pillars 7 and gaps 8extending the entire distance to the base 5. In general, reducing thedepth of any gaps in the partitions can increase the electricalisolation between compartments 4 and reduce the tendency for offsetcurrents between the electrodes 13 of different compartments 4, forexample if the apolar medium becomes hydrated sufficiently to provide anelectrical conductivity path between electrodes 13. Apart from thealternative constructions of the partitions 6, supports 2 otherwise havethe same construction as described above.

A first alternative construction for the partitions is shown in FIGS. 12and 13 and arranged as follows. In the first alternative construction,the partitions 6 have no gaps allowing the flow of apolar medium betweenthe compartments 4. In particular, the partitions 6 have recesses 30that define the compartments 4 without gaps between those compartments4. The partitions 6 may be formed by a common body 31 extending from thebase 5. In that case, the base 5 has planar surfaces forming the innerends of the compartments 4. The common body 31 may be formed as aseparate layer laminated with the base 5, but alternatively may beintegral with the base 5 and the recesses 30 formed by removingmaterial.

In this example, the partitions 6 have a profile as viewed across thesupport 3 that is the same as the profile of the inner portion 20 of thepartitions in the second alternative construction. That is, the profileis undulating and comprises, around individual compartments 4, pluralsalient portions 32 that protrude into the compartment 4 and pluralre-entrant portions 33 where the compartment 4 protrudes into thepartitions 6.

The salient portions 32 are arranged physically to constrain a volume 2of polar medium inside the compartment 4. Thus, the dimensions of thesalient portions 32 control the size of the volume 2 of polar mediumthat may be accommodated in the compartment 4.

The re-entrant portions 33 provide channels that extend outside a volume2 of polar medium accommodated in the compartment 4. Therefore, there-entrant portions 33 allow outflow of apolar medium displaced by entryof a volume 2 of polar medium into the compartment 4.

This undulating structure also reduces the surface area of thepartitions 6 that is in contact with a volume 2 of polar medium. Thisserves to allow the a volume 2 of polar medium to move to the base ofthe compartment 4 and thereby assist in making electrical contact withthe electrode 13.

In principle, any number of re-entrant portions 33 could in principle beprovided such as 3, 4, 5, 6 etc. However one would need to balance thenumber of salient portions 32 with the contact surface for the volume 2of polar medium.

The salient portions 32 as shown in FIG. 12 have rounded edges.Alternatively the salient portions 32 may have sharp edges. Such sharpedges may reduce further the extent of contact between the edge of thecompartment 4 and the volume 2 of polar medium. Conversely, sharp edgesmay puncture the layer of amphiphilic molecules. It is advantageous toreduce the extent of contact of the volume 2 of polar medium with theinner surface of the compartment 4. Having salient portions 32 enableslarger volumes 2 of the polar medium to be used for a given volume ofcompartment 4.

As can be seen from FIG. 12, the dimensions and shape of the re-entrantportions 33 determines the surface area which is capable of beingcontacted by a volume 2 of polar medium. The salient portions 32 andre-entrant portions 33 are interrelated in that generally the greaterthe cross-sectional width of the re-entrant portion 33, the greater thereduction in surface area of the walls of the compartment 4.

Thus, compared to the constructions described above, in the firstalternative construction, the partitions 6 provide the same function ofconstraining the volumes 2 of polar medium and preventing them fromcontacting or merging, but the electrical isolation between compartments4 is increased due to the absence of gaps in the partitions 6. Theabsence of gaps in the partitions 6 also reduces the beneficial effectof allowing flow of apolar medium between compartments 4, but this is tosome extent mitigated when filling compartments 4 by the re-entrantportions 33 providing channels allowing outflow of displaced apolarmedium which assists insertion of a volume 2 of polar medium. Thisallows for a volume 2 of polar medium of maximum size to be inserted,the movement of which is constrained by the salient portions 32.

The partitions 6 have the same height so that the outer ends 34 of thepartitions 6 extend in a common plane, as shown in FIG. 12, to providethe support 3 with a brush-like planar upper surface.

There will now be described some alternative constructions for thepartitions 6 in which the partitions 6 comprise inner portions defininginner recesses of the compartments without gaps therebetween, and outerportions extending outwardly from the inner portions defining outerportions of the compartments with gaps allowing the flow of apolarmedium between the compartments. Thus, effectively the gaps extendpartway to the base 5. Apart from the alternative constructions of thepartitions 6, the following supports 2 otherwise have the sameconstruction as described above.

A second alternative construction for the partitions is shown in FIGS.14, 15 and 16 and arranged as follows.

The apparatus 1 for holding an array of volumes 2 of a polar medium inapolar medium comprises a support 3 providing an array of compartments4. In use, all the compartments 4 contain apolar medium, and at leastsome of the compartments 4 contain single volumes 2 of a polar medium inthe apolar medium.

The support 3 comprises a base 5 and partitions 6 that extend from thebase 5. Herein, the terms “inner” and “outer” describe relativelocations within the compartments 4 from the openings at the outer endtowards the base 5 at the inner end.

As described in more detail below, the partitions 6 define compartments4 having openings provided at the distal ends of the partitions 6. Theseopenings provide communication from the compartments 4 into the spaceadjacent the support 3, and volumes of polar medium may be introducedinto the compartments 4 through the openings. The compartments 4 arearranged such that the volumes 2 of polar medium are physicallyseparated from each other. This prevents the volumes 2 of polar mediumfrom merging or contacting each other to form interfaces. This providesa very stable array of volumes 2 of polar medium which is capable ofbeing stored over a long period of time.

Optionally, a dam (taking the form shown in FIG. 1) may be providedaround the perimeter of the support 3 which aids in filling theperipheral edges of the support 3 with apolar medium. One or morechannels may be provided in the dam through which apolar medium may beintroduced or drained from the support 3.

The support 3 may be prepared from a range of different materials havinga high electrical resistance, including without limitation undopedcrystalline silicon (i.e. a silicon wafer), SUB, polycarbonate, and/orpolyester, and including any combination of these or other materials.The support 3 may be manufactured using conventional techniques for suchmaterials, including, without limitation, deposition and removaltechniques for example etching or laser processing.

The base 5 comprises a substrate 12. The substrate 12 supports anelectrode 13 in each compartment 4. In this example, the electrodes 13are shown recessed into the substrate 12, but they could alternativelybe deposited as an outer layer on an exposed surface of the substrate12. The electrodes 13 are provided to make electrical contact with thevolumes 2 of polar medium contained in the compartments 4 and arediscussed in more detail below.

The substrate 12 may optionally comprise a surface coating. The surfacecoating may provide a high resistance outer layer. One possiblecombination of materials for the base 5 is that the base 5 is made ofundoped crystalline silicon (i.e. a silicon wafer) and the coating to bemade of SUB. Such a surface coating may be provided on top of thesubstrate 12 with apertures aligned with the electrodes 13 to allowelectrical contact between the electrodes 13 and the volumes 2 of polarmedium. As an alternative, the electrodes 12 could be patterned in thesame layer as the surface coating or on top of the surface coating.

The partitions 6 may be made of the same or different material to thebase 12 of the support 3 and may have the same or different surfaceproperties. The partitions 6 are typically apolar and may be made forexample from Permex. The partitions 6 may optionally comprise a surfacecoating (not shown) to modify their electrical and/or physicalproperties.

FIGS. 15 and 16 show a particular arrangement of the partitions 6, butthis is is not essential and the partitions 6 may have a variety ofdifferent arrangements to define the compartments 4 so as to constrainthe volumes 2 of polar medium in the compartments 4 from contactingvolumes 2 comprising polar medium in neighbouring compartments.

In the arrangement of FIGS. 15 and 16, the partitions 6 comprise innerportions 20 and outer portions 21.

The inner portions 20 of the partitions 6 define inner recesses 22 thatform the inner portions of the compartments 4 without gaps between thoseinner portions of the compartments 4. The inner portions 20 of thepartitions 6 may be formed by a common body extending from the base 5.In that case, the base 5 has planar surfaces forming the inner ends ofthe compartments 4. The inner portions 20 may be formed as a separatelayer laminated with the base 5 after removal of material to formapertures that become the inner recesses 5. Alternatively the innerportions 20 may be integral with the base 5 and the recesses 22 formedby removing material of the integral member.

In this example, the inner portions 20 of the partitions 6 have aprofile as viewed across the support 3 that is circular aroundindividual compartments 4.

The outer portions 21 of the partitions 6 extend outwardly from theinner portions 20 and define the outer portions of the compartments 4.In this example, the outer portions 21 of the partitions 6 compriseplural pillars 23 that extend out from the inner portions 20 of thepartitions 6 as shown in FIG. 15, in this example perpendicularly, witha similar pattern to the pillars 7 in the construction of the partitions6 shown in FIGS. 4 and 5. In particular, a cross-shaped pillar 23 a inthe corners of the compartments 4 with arms extending towards thecompartment 4 and plural further pillars 23 b along the each side of thecompartment 4, with gaps 24 between the cross-shaped pillars 23 a andthe further pillars 23 b, and between the further pillars 23 b.

The pillars 23 have gaps 24 therebetween. In this example, the gaps 24extend to the inner portions 20 of the partitions and hence only partwayto the base 5. The gaps 24 are of sufficient size to allow the flow ofan apolar medium between the compartments 4, whilst maintaining theseparation of the volumes 2 of polar medium in the compartments 4. Theprovision of gaps 24 allows the apolar medium to flow between thecompartments 4. This aids in filling of the compartments 4 as apolarmedium may be displaced by a volume 2 of polar medium entering acompartment 4. Further description of this is given below. The gaps 24also allows the level of apolar medium in the support 3 to be controlledand equalised across the array. Thus, compared to the constructionsdescribed above, in the second alternative construction, the partitions4 provide the same function of constraining the volumes 2 of polarmedium and preventing them from contacting or merging, and the gaps 24provide the same function to the gaps 8 of allowing flow of apolarmedium between compartments 4. However, the electrical isolation betweencompartments 4 is increased due to the absence of gaps in the innerportions 20.

The pillars 23 are set back from the edges of the inner recesses 22 asviewed from the openings of those inner recesses 22. This creates a stepon the upper surface of the inner portions 20 of the partitions 6between any given pillar 23 and the adjacent inner recesses 22.

The pillars 23 have the same height so that the outer ends 25 of thepillars 24 extend in a common plane, as shown in FIG. 15, to provide thesupport 3 with a brush-like planar upper surface.

The relative heights of the inner portions 20 and outer portions 21 ofthe partitions 6 may be varied. In one typical embodiment, the innerportions 20 have a height of 90 μm and a diameter of 170 μm, and outerportions 21 have a height of 60 μm.

In this example, the inner portions of the partitions further comprisetwo re-entrant portions 28. As can be seen from FIGS. 15 and 16, thedimensions of the re-entrant portions are relatively small compared tothe inner surface of compartment 4.

A modified construction for the partitions is shown in FIGS. 17 and 18.This is similar to the construction of FIGS. 15 and 16 except for thefollowing modifications.

Firstly, the inner recesses 22 formed by the inner portions 20 of thepartitions 6 have a profile as viewed from the openings of thecompartments 4 across the support 3 that is not circular. In particular,the profile is undulating and comprises, around individual compartments4, plural salient portions 26 that protrude into the compartment 4 andplural re-entrant portions 27 where the compartment 4 protrudes into thepartitions 6.

The salient portions 26 are arranged physically to constrain a volume 2of polar medium inside the compartment 4. Thus, the dimensions of thesalient portions 26 control the size of the volume 2 of polar mediumthat may be accommodated in the compartment 4.

The re-entrant portions 27 provide channels that extend outside a volume2 of polar medium accommodated in the compartment 4. Effectivelytherefore, the inner portions 20 of the partitions 6 have surfaces thatare indented with a plurality of channels that extend outwardly of theinner recesses 22. Therefore, the re-entrant portions 27 allow outflowof apolar medium displaced by entry of a volume 2 of polar medium intothe compartment 4.

This undulating structure also reduces the surface area of thepartitions 6 that is in contact with a volume 2 of polar medium. Thisserves to allow a volume 2 of polar medium to move to the base of thecompartment 4 and thereby assist in making electrical contact with theelectrode 13.

In principle, any number of re-entrant portions 27 could in principle beprovided such as 3, 4, 5, 6 etc. However one would need to balance thenumber of salient portions 26 with the contact surface for the volume 2of polar medium.

Secondly, the pillars 23 of the outer portions 21 of the partitions 6have a different pattern. In particular, a cross-shaped pillar 23 c inthe corners of the compartments 4 with arms extending in a directionalong the side of the compartment 4 and a pair of further pillars 23 calong the each side of the compartment 4, with gaps 24 between thecross-shaped pillars 23 c and the further pillars 23 d, and between thefurther pillars 23 d. This is enable the pillars 23 to fit on the innerportion 20, but the pillars 23 have the same function and effect.

The salient portions 26 as shown in FIG. 17 have rounded edges.Alternatively the salient portions 26 may have sharper edges. It isadvantageous to reduce the extent of contact of the volume 2 of polarmedium with the inner surface of the compartment 4. Having salientportions 26 enables larger volumes 2 of the polar medium to be used fora given volume of compartment 4.

As can be seen from FIG. 17, the dimensions and shape of the re-entrantportions 27 determines the surface area which is capable of beingcontacted by a volume 2 of polar medium. The salient portions 26 andre-entrant portions 27 are interrelated in that generally the greaterthe cross-sectional width of the re-entrant portion 27, the greater thereduction in surface area of the walls of the compartment 4.

A modified construction for the partitions 6 is shown in FIGS. 19 and20. This is similar to the construction of FIG. 15 except for amodification that the surfaces 64 of the inner recesses 22 and thesurfaces 66 of the pillars 23 have a patterning described further below.

A yet further modified construction for the partitions 6 is shown inFIGS. 21 and 22. This is the same as the construction of FIG. 19 exceptfor the size of the patterning.

A yet further modified construction for the partitions 6 is shown inFIGS. 23 and 24. This is the same as the construction of FIG. 15 exceptfor a modification that the surfaces 64 of the inner recesses 22 (butnot the surfaces 66 of the pillars 23) have a patterning as describedbelow.

A yet further modified construction for the partitions 6 is shown inFIGS. 25 and 26.

The patterning on the various surfaces of the compartments 4 shown inFIGS. 19 to 26 will now be described in more detail.

In particular, the surfaces 64 of the inner recesses 22 are indentedwith a plurality of indentations 65 that extend outwardly of the innerrecesses 22, and hence outwardly of the compartments 4, along the entirelength of inner recesses 22. In this example, the indentations 65 arerectangular in cross-section. Similarly, surfaces 66 of the pillars 23are indented with a plurality of indentations 67 that extend outwardlyof the compartments 4 (except in the construction of FIG. 15). In thisexample, the indentations 67 are rectangular in cross-section.

The surfaces 64 of each inner recesses 22 between the indentations 65lie in a common curved plane extending around the inner recess 22. Thesesurfaces 64 physically constrain a volume 2 of polar medium inside theinner recess 22. Thus, the dimensions of the surfaces 64 control thesize of the volume 2 of polar medium that may be accommodated in theinner recess 22.

The indentations 65 hold polar medium that reduces the surface area ofthe partitions 6 that is in contact with a volume 2 of polar medium.This modifies the surface properties of the pillars 7, repelling polarmedium and therefore assisting in constraining a volume 2 of polarmedium held in the inner recess 22, and in allowing entry of the polarmedium into the inner recess 22. In general, the patterning couldcomprise other surfaces features to achieve this effect.

An initial pre-treatment of apolar medium 70 is applied as describedbelow. The indentations 65 and 67 assist in spreading the pre-treatmentof apolar medium 70 by wicking it over the substrate 3.

The pre-treatment of apolar medium 70 added to the partitions 6 is heldwithin the indentations 65 by surface tension/capillarity which servesto increase the phobicity of the partitions 6 to the polar medium andtherefore the contact angle between the volume 2 of polar medium and thepartitions 6. This helps define the shape of the meniscus of the volume2 of polar medium. Indentations 65 having a high capillarity arepreferred as they retain the apolar medium more effectively and preventor hinder flow of apolar medium onto the surface of the electrode 12.Thus polar medium added subsequently to the compartments is able todirectly contact the electrodes 13.

The indentations 65 and surfaces 64 have widths according to anembodiment preferably of at most 20 μm, more preferably of at most 10μm. The indentations 65 and surfaces 64 have widths that is smallcompared to the size of the volume of volume 2 of polar medium held inthe inner recess 22. For example, if the dimensions of the inner recess22 are characterised with reference to the diameter d of the largestnotional sphere that can be accommodated within the inner recess 22,then the indentations 65 and surfaces 64 have widths that are preferablyat most 0.1d, more preferably at most 0.05d. By way of example, wherethe inner recess 22 has a depth of 90 μm, the outer portions 23 have aheight of 30 μm and the diameter d is 140 μm, the indentations 65 andsurfaces 64 may have widths that are 5 μm. Similarly, in theconstruction of FIG. 19, the indentations 65 and surfaces 64 have widthsthat are 5 μm.

The depth of the indentations 65 is chosen to allow the channels toretain the polar medium. By way of example, in the construction of FIG.19, the indentations 65 have a depth of 5 μm, providing an aspect ratioof 1:1.

However, deeper indentations 65 provide more effective capture andretention of polar medium. By way of example, in the construction ofFIG. 25 and FIG. 21, the indentations 65 have a depth of 50 μm,providing an aspect ratio of 10:1. This captures and retains oil moreeffectively within the channels due to higher capillarity. The availabledroplet diameter d is 100 μm. An added benefit of the higher aspectwells is that they provide a smaller droplet diameter which in turnprovides a smaller amphipathic membrane area.

The surfaces 66 of the pillars 23 between the indentations 67 lie in acommon curved plane extending around the inner recess 22. Theindentations 67 hold polar medium which repels the apolar medium. Thepre-treatment of apolar medium 70 added to the partitions 6 is heldwithin the indentations 65 by surface tension/capillarity which servesto increase the phobicity of the partitions 6 to the polar medium andthereby assists in the filling of the inner recess 22.

The indentations 67 and surfaces 66 have widths preferably of at most 20μm, more preferably of at most 10 μm. The indentations 67 and surfaces66 have widths that is small compared to the size of the volume ofvolume 2 of polar medium held in the inner recess 22. For example, ifthe dimensions of the inner recess 22 are characterised with referenceto the diameter d of the largest notional sphere that can beaccommodated within the inner recess 22, then the indentations 65 andsurfaces 64 have widths that are preferably at most 0.1d, morepreferably at most 0.05d. By way of example, where the inner recess 22has a depth of 90 μm, the outer portions 23 have a height of 30 μm andthe diameter d is 140 μm, the indentations 65 and surfaces 64 may havewidths that are 5 μm. However, deeper indentations 67 provide moreeffective retention of polar medium, but it is difficult to providehigher aspect indentations 67 due to the limited space.

The following comments apply to the support 3 with any of theabove-described constructions.

The support 3 may comprise any number of compartments 4. The support 3may comprise, for example, number of compartments 4 in the range from 2to 10⁶, but may typically be in the range from 100 to 100,000.

An individual compartment 4 has a notional cross-sectional area definedby the spacing between the partitions 6 and a notional volume defined bythe height of the partitions 6. The notional volume is typically thesame for all compartments 4 of the array.

As in the examples above, the compartments 4 may have irregularly shapedperipheries as viewed across the support 3. Irrespective of the shape ofthe compartments 4, in the case where a compartment contains a singlevolume of the polar medium, the dimensions of a compartment 4 may becharacterised with reference to the largest notional sphere that can beaccommodated within the compartment 4. That is approximately the size ofthe largest volume 2 of polar medium that could be accommodated in thecase that the volumes 2 of polar medium are spherical (which is notessential). Indeed, in the case where the volumes of polar medium areliquid, they can deform depending upon the dimensions of the compartmentand the surface properties of the support. Such a size may typically bebetween 50 μm and 500 μm, more typically between 70 μm and 200 μm

The array will typically contain volumes 2 of polar medium of asubstantially similar size.

The dimensions of the compartment 4 may be chosen depending upon thesize of the volumes 2 of polar medium to be contained. The volumes 2 ofpolar medium typically have an average diameter in the range from 5 μmto 500 μm or an average volume in the range from 0.4 pL to 400 nL. Thedensity of the compartments 4 in the support 3 is therefore dependentupon the size of the volumes 2 of polar medium and the particulararrangement of the partitions 6.

In the above examples, the partitions 6 have a regularly repeatingpattern so that the compartments 4 have the same size and shape acrossthe support 3 and are arranged in a regular array. This is notessential. The partitions 6 and compartments 4 may have alternativelyhave differing shapes and/or sizes across the support 3 and/or thecompartments 4 may be arranged in an irregular array.

The nature of the polar medium of the volumes 2 of polar medium is asfollows.

The polar medium may be a hydrophilic medium. The hydrophilic medium mayfor example comprise an aqueous medium.

In one example, the polar medium of the volumes 2 is an aqueous buffersolution. The buffer solution may comprise a supporting electrolyte.

The array may be filled with an emulsion or filled with volumes ofapolar and polar volumes by use of a flow-cell assembly such as shown inFIG. 27. In FIG. 27, an array 101 attached to an ASIC/PCB 105 isinserted into the array retainer 102. A protective gasket 107 is placedon the surface of the array and the array is affixed to the fluidicmodule 103 using screws 109. Fluid may be flowed over the surface of thearray in order to fill the compartments. Valve rotor 110 may be rotatedin order to fluidically seal the flow cell. Fluid enters the flow-cellfrom a fluid reservoir (not shown) and exits the flow cell, as shown bythe arrows.

In an example where the volumes 2 are pre-formed before being disposedin the compartments, the volumes 2 may be droplets of an aqueous buffersolution. In that case, they may be made in conventional manner, forexample using a microfluidic flow T-junction 40 as shown in FIG. 28comprising a first flow channel 41 containing the polar medium and asecond flow channel 42 comprising the apolar medium. The two flowchannels 41 and 42 intersect at the T-junction spontaneously formingdroplets 43 which flow downstream from the T-junction and may becollected in a vessel 44 as an emulsion of the droplets 43 in the apolarmedium. The size of the droplets 43 is determined by the flow rates ofthe polar and apolar fluids as well as the width of the apertures of therespective flow channels 41 and 42. FIG. 28 also shows droplets 43 thathave been formed by the T-junction 40.

Droplets may be provided having different amounts of substances, by forexample providing a third flow channel containing a different polarmedium to the first flow channel which intersects with the first channelto form a common flow channel prior to intersecting at the T-junction.The flow rates of the third and first flow channels may be varied toprovide droplets having varying ratios of components.

In another example where the volumes 2 are pre-formed before beingdisposed in the compartments, the volumes 2 of polar medium may be beadsof an aqueous gel, such as an agarose gel. The gel may comprise anaqueous buffer solution as the liquid phase. The buffer solution maycomprise a supporting electrolyte. Examples of such are non-crosslinkedor crosslinked hydrogels such as agarose or sepharose. A bead may beformed in-situ from a droplet for example by cooling or crosslinkingwith UV. A bead introduced into the apolar medium may form a droplet,for example by melting. The volume of polar medium may be providedwithin a porous plastic or glass bead.

Where the volumes 2 of polar medium are beads of an aqueous gel, theymay have sufficient rigidity to protrude out of the compartments. FIG.29 shows an apparatus that is an example of this. In this example, thebead protrude above the height of the partitions 6 and the meniscus 52is formed as shown.

It may be the case that, when the volumes 2 of polar medium are beads ofan aqueous gel, the leakage currents between the compartments 4 isreduced. Gel beads can be made in a conventional manner in T-piecedroplet maker by merging a stream liquid gel at an elevated temperatureinto a stream of the apolar medium and allowing to cool, thereby to forman emulsion of beads of gel in the apolar medium. Gel beads may also beeasier to locate onto a spiked electrode in the well and are generallymore dimensionally stable.

In the case of using gels, shapes other than spherical may be created,for example elongate cigar shaped structures which might be employed indeep recesses (thus maximising the internal volume of the volume 2 ofpolar medium). This would have the advantage of extending the lifetimeof the volume 2 of polar medium for example if the redox mediator werecontained within the volume 2 of polar medium.

The aqueous gel may be a cross-linked gel. These are gels in which thematrix is cross-linked, which increases the hardness of the gel,providing a higher structural integrity than gels that are notcross-linked. For example, agarose gels may be cross-linked. Beads ofcross-linked gel are commercially available and may be mixed with apolarmedium to form an emulsion of beads of gel in the apolar medium. Onepossibility is cross-linked agarose beads with a particle size of 160 μmand an agarose content of 6.8-7.2% (as available for example fromWorkBeads™ 200SEC, BioWorks), which are highly porous and physicallystable. The beads may be supplied from the manufacturer and may becoated with an amphipathic layer by introducing the beads into an apolarmedium containing amphipathic molecules. This also permits an easiermethod of manufacture of such volumes 2 of polar medium.

Cross-linked gels may also provide advantages in inserting volumes 2 ofpolar medium into compartments 4 during manufacture the apparatus 1 asdescribed below.

Although in the above examples, a single volume 2 of polar medium iscontained in an individual compartment, as an alternative plural volumes2 of polar medium may be contained in a compartment 4. As an example ofthis, FIG. 30 shows an apparatus 1 in which two volumes 2 of polarmedium are provided within a single compartment 4. The volumes 2 ofpolar medium are positioned on top of each other and may have a furtherlayer 50 comprising polar medium provided in contact with one of thevolumes 2 of polar medium. An membrane comprising amphipathic moleculesmay be provided at any interface between volumes 2 of polar medium, aswell as at the interface between one of the volumes 2 of polar mediumand the layer 50 of polar medium. Ion channels may also be provided inany such membranes. Provision of plural volumes 2 of polar medium in acompartment 4, for example as shown in FIG. 30, may increase theeffective amount of the polar medium relative to the volume of thecompartment 4. This provides advantages such as enabling a larger amountof mediator to be provided.

The nature of the apolar medium is as follows.

The apolar medium may be a hydrophobic medium.

The apolar medium may comprise a hydrocarbon or an oil or a mixturethereof. Suitable oils include silicone oil, AR20 or hexadecane. Theapolar medium may be substantially immiscible with the polar medium ofthe volumes 2.

The apparatus 1 holding the array of volumes 2 of polar medium in asupport 3, as described above, may have a wide range of biological,pharmaceutical and other analytical applications. It provides theopportunity to facilitate high throughput processing of small volumes 2or groups of volumes 2 and may be used for example to compartmentalisereactions, cell sorting and screening applications such as proteincrystallisation, analysis of blood or spinal fluid and waste processing.The ability to address and replace the volumes 2 of polar medium in thearray is an important aspect, for example for carrying out reactions onthe volumes 2 and replenishing the array.

In some applications, the apparatus 1 holding the array of volumes 2 ofpolar medium in a support 3, as described above, may be provided with alayer 50 of a polar medium as shown in FIG. 31 (which illustrates by wayof example the case that the volumes 2 of polar medium are droplets inan apolar medium). The layer 50 of a polar medium extends across thesupport 3 over the openings of the compartments 4. Thus the layer 50 ofa polar medium rests on the partitions 6. The layer 50 of a polar mediumis also in contact with at least some of the volumes 2 of polar mediumpreferably all of them. Membranes comprising amphipathic molecules areformed at the interfaces 51 between the layer 50 of polar medium and thevolumes 2 of polar medium.

In order to form the membranes comprising amphipathic molecules, theamphipathic molecules may initially be provided in any one of more ofthe volumes 2 of polar medium, the layer of apolar medium or the layer50 of a polar medium. In any of these cases, the membranes may form whenthe layer 50 of polar medium is flowed across the support 3. In the caseof the amphipathic molecules being provided in the volumes 2 of polarmedium, the volumes 2 of polar medium disposed within the compartments 4may comprise a layer of amphipathic molecules around the surfacesthereof prior to provision of the layer 50 of a polar medium. In thecase of the amphipathic molecules being provided in the layer 50 of apolar medium, the layer 50 of a polar medium may comprise a layer ofamphipathic molecules on the surface that is brought into contact withthe volumes 2 of polar medium.

The membranes comprising amphipathic molecules form at the at theinterfaces 51 when the layer 50 of polar medium and the volumes 2 ofpolar medium are brought into contact. The membranes comprisingamphipathic molecules separate the layer 50 of polar medium and thevolumes 2 of polar medium.

The polar medium of the layer 50 may be the same or different materialas the volumes 2 of polar medium. The polar medium of the layer 50 maybe a hydrophilic medium. The hydrophilic medium may for example comprisean aqueous medium. In one example, the hydrophilic medium of the layer50 comprises an aqueous buffer solution. The buffer solution maycomprise a supporting electrolyte.

The nature of the amphipathic molecules is as follows.

The amphipathic molecules may be of any type that is capable of forminga membrane at the interfaces 51 between the layer 50 of polar medium andthe volumes 2 of polar medium.

The method and apparatus of the invention is suitable for use withnumerous different types of amphipathic molecules.

In one example, the amphipathic molecules may comprise a lipid, whichmay have a single component or a mixture of components, as isconventional when forming lipid bilayers.

Any lipids that form a lipid bilayer may be used. The lipids are chosensuch that a lipid bilayer having the required properties, such assurface charge, ability to support membrane proteins, packing density ormechanical properties, is formed. The lipids can comprise one or moredifferent lipids. For instance, the lipids can contain up to 100 lipids.The lipids preferably contain 1 to 10 lipids. The lipids may comprisenaturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety andtwo hydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester.

The lipids can also be chemically-modified. The head group or the tailgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000]; functionionalised PEG Lipids, such as1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000]; and lipids modified for conjugation, such as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and1,2-Dipalmitoyl-snGlycero-3-Phosphoethanolamine-N-(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. Examplesof suitable lipids include without limitation phytanoyl lipids such as1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE). However suchnaturally occurring lipids are prone to biological degradation forexample by proteins or detergents and are not able to withstand highvoltages. Preferably the amphipathic layer is non-naturally occurring.Amphipathic polymer membranes are preferred over lipid membranes due totheir ability to withstand higher voltages.

In another example, the amphipathic molecules may comprise anamphipathic compound comprising a first outer hydrophilic group, ahydrophobic core group, and a second outer hydrophilic group, whereineach of the first and second outer hydrophilic groups is linked to thehydrophobic core group.

Some such amphipathic compounds are disclosed in the InternationalPatent Application filed on the same day as this application entitled“Droplet Interfaces” [ONT Ref: ONT IP 039] which is incorporated hereinby reference.

Other such amphipathic compounds are disclosed in U.S. Pat. No.6,916,488 which is incorporated herein by reference and discloses anumber of polymeric materials that can be employed in the apparatus 1 asplanar amphipathic membranes. In particular triblock copolymers aredisclosed, for example silicon triblock copolymer membranes such aspoly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyloxazoline)(PMOXA-PDMS-PMOXA).

The use of such triblock copolymers as amphipathic membranes in thepresent invention is particularly preferred due to their ability towithstand high voltages, their robustness as well as their ability towithstand biological degradation from detergents and proteins. Theirability to withstand biological degradation allows the directapplication and measurement of biological samples to the array, such asfor example blood or serum. The polar layer applied to the top surfacemay be the sample to be determined. Examples of silicone triblockpolymers that may be employed are 7-22-7 PMOXA-PDMS-PMOXA, 6-45-6PMOXA-PE-PMOXA and 6-30-6 PMOXA-PDMS-PMOXA, where the nomenclaturerefers to the number of subunits. For example, 6-30-6 PMOXA-PDMS-PMOXAis comprised of 30 PDMS monomer units and 6 PMOXA monomer units.

Depending on the nature of the amphipathic molecules, the membranes maybe bilayers of the amphipathic molecules or may be monolayers of theamphipathic molecules.

Some possible methods of forming an array of volumes 2 in the apparatus1 are as follows.

First, there is provided the apparatus 1 comprising the support 3arranged as described above.

In a first type of method, the volumes 2 of polar medium are pre-formedin the apolar medium before disposition in the compartments. There willnow be described an example of this type of method in which first anemulsion of the volumes 2 of a polar medium in an apolar medium is madeusing the methods mentioned above.

The amphipathic molecules may be provided to the volumes 2 of a polarmedium or the apolar medium. This may be achieved simply by adding theamphipathic molecules to the emulsion and whereupon they migrate to theinterfaces between the volumes 2 of a polar medium and the apolarmedium. Alternatively the amphipathic molecules may be added to theapolar medium prior to forming the emulsion.

To dispose the polar medium and apolar medium on the support 3, theemulsion is flowed over the support 3. This has the effect that theapolar medium flows into the compartments 4 and respective volumes 2 ofpolar medium within the apolar medium further flow into at least some ofthe compartments 4 through the openings. This has been found to occurnaturally as the emulsion flows over the upper surface of the support 3,assisted by the design of the support 3 as describe above. The apolarmedium and volumes 2 of polar medium are drawn into the array bycapillary forces. In addition in supports 2 having gaps betweencompartments 4, the apolar medium flows between compartments through thegaps.

The emulsion typically contains more volumes 2 of polar medium than thenumber of compartments to ensure that a relatively large proportion ofthe compartments 4 are populated with volumes 2 of polar medium. Theexcess volumes 2 of a polar medium may be removed by washing the support3 with the apolar medium. The washing leaves volumes 2 of polar mediumin the compartments and leaves a layer of the apolar medium used forwashing as a layer of apolar medium extending across the openings incontact with the volumes 2 of polar medium.

In this method, the emulsion may further comprise the amphipathicmolecules. This facilitates the formation of the membranes when polarmedium is flowed across the openings in the support to form a layercomprising polar medium, as described below. The presence of theamphipathic molecules also stabilises the emulsion.

The relative viscosities of the polar medium of the volumes 2 and apolarmedium may be selected to be sufficiently similar that volumes 2 of apolar medium does flowing the emulsion over the support 3 do not floatat the surface of the apolar medium away from the support 3. It is notedhowever that typically the volumes 2 of polar medium are drawn and heldwithin the compartments 4 by capillary forces such that even if anapolar medium of a higher density than the volumes 2 of polar medium isused, the volumes 2 of a polar medium tend to remain within the apolarmedium at the electrode surface.

This method also intrinsically provides a layer comprising apolar mediumthat extends across the openings of the compartments 4 in contact withthe volumes 2 of polar medium in the compartments 4, being the apolarmedium of the emulsion, or the apolar medium used to wash the support 3.

A dye may be incorporated into the volumes 2 of polar medium such thatthe presence of droplets in the array may be more easily visualised. Acoloured dye, preferably of a different colour to that added to thevolumes 2 of polar medium may be added to the apolar medium to moreeasily visualise the distribution of the apolar medium across thesupport 3. The incorporation of dyes within the volumes 2 of polarmedium and/or apolar medium may be employed as a quality control checkduring fabrication to ensure that the compartments 4 are sufficientlypopulated with volumes 2 of polar medium and/or the apolar medium isproperly distributed.

When the volumes 2 of polar medium are beads of an aqueous cross-linkedgel, the emulsion may be flowed over the support 3 under positivepressure. This is possible because the cross-linked gels are harder andable to withstand the pressure, which is chosen having regard to themechanical properties of the cross-linked gel. In contrast, beads of geland droplets of solution can have a greater tendency to deform and mergeunder pressure. The use of such a positive pressure assists in fillingof the compartments 4. This is particular advantageous when using asupport with the first alternative construction or other constructionswithout gaps between the compartments, which are in general terms harderto fill.

In another example of the first type of method in which the volumes 2 ofpolar medium are pre-formed in the apolar medium, the volumes comprisingpolar medium may be dispensed directly into individual compartments, forexample by acoustic droplet injection. With this technique, thedispensing may be controlled such that the correct number of volumescomprising the polar medium are dispensed without the need to removeexcess volumes. In one embodiment of this technique, the substrate 3comprises compartments 4 without gaps in the partitions, in which caseit is desirable that the width of the volumes 2 of polar medium is lessthan the width of the opening of the compartment 4. The volume 2 mayconsist of a polar medium or comprise a polar medium within an apolarmedium. In another embodiment, the substrate 3 comprises compartments 4having gaps 8 in the partitions 6, wherein the gaps 8 extend fully fromthe openings to the base 5 of the support 3. In a yet furtherembodiment, the substrate 3 comprises compartments 4 having gaps 8 inthe partitions 6, wherein the gaps may extend partially from theopenings to the base 5. In the case that the gaps extend fully from theopenings to the base of the support, a pretreatment may beadvantageously added to the support prior to the addition of the volumesin order to constrain the droplets and prevent them from merging.

In a second type of method, the volumes 2 of polar medium are formed inthe compartments 4 from a larger amount of polar medium that is flowedinto the cell. Examples of such methods will now be described withreference to the schematic flow diagrams of FIGS. 32 to 34 which showthe support 3 in successive steps of the method. In FIG. 32, the support3 is of the type described above in which the partitions 6 compriseinner portions 20 defining inner recesses 21 without gaps, and outerportions 21 with gaps 23 In FIG. 32, the support 3 is illustratedschematically, and could for example be any of the second to eleventhalterative constructions described above.

First the support 3 is provided as shown in FIG. 32( a).

The support 3 is pre-treated with a pre-treatment apolar medium 70 asshown in FIG. 32( b). The pre-treatment apolar medium 70 may be of thesame or different material from the layer of apolar medium subsequentlyapplied as described below.

The pre-treatment apolar medium 70 (which may be diluted in a solvent)is added to the substrate 3 (for example by pipette) and allowed tospread across the substrate by capillarity. The pre-treatment apolarmedium 70 collects inside the corners of the inner recess 22 and aroundthe pillars 23 of the outer portions 21, in particular in the cornersbetween the pillars 23 and the upper surface of the inner portion 20.

Next, polar medium 71 and apolar medium 74 are disposed on the support 3as follows.

Polar medium 71 is flowed across the support 3 so that the polar medium71 enters into the compartments 4 through the openings, as shown in FIG.32( c). One way of doing this is to attach one end of the apparatus 1 toa flow cell 60. At least a portion of the are of the electrode 13 isfree from apolar medium and therefore the volume 2 of polar medium makeselectrical contact with the electrode 13.

In contrast to the first type of method wherein the volumes 2 of polarmedium and the apolar medium are disposed on the support 3 together, forexample in an emulsion, herein the layer of apolar medium is providedsubsequently.

Excess polar medium 71 is removed by flowing a displacement fluid havinga different phase from the polar medium across the substrate 3, leavingthe volumes 2 comprising polar medium in the compartments 4. Twoalternative approaches for this are described.

The first approach is illustrated in FIGS. 32( d) and (e). In the firstapproach, the displacement fluid is apolar medium 74 which is flowedacross the substrate 3 as shown in FIG. 32( d). Clipping of the polarmedium 71 takes place at the outer edge of the inner portion, as shownin FIG. 32( e). Relaxation of the volume of polar medium takes place asshown by FIG. 32( j) to leave the volumes 2 comprising polar medium inthe compartments 4. This first approach leaves a layer 73 comprisingapolar medium extending across the openings of the compartments 4 incontact with the volumes 2 comprising polar medium.

The second approach is illustrated in FIGS. 32( f) to (i) which stepsoccur instead of FIGS. 32( d) and (e). In the second approach, thedisplacement fluid is a gas 72 which is flowed across the substrate 3 asshown in FIG. 32( f). Clipping of the polar medium 71 takes place at theouter edge of the inner portion, as shown in FIG. 32( g), leaving thevolumes 2 comprising polar medium in the compartments 4 and a layer ofthe gas 72 extending across the openings of the compartments 4 incontact with the volumes 2 comprising polar medium, as shown in FIG. 32(h). The gas 72 is preferably inert, and may be air or any other gas.

Thereafter, apolar medium 74 is flowed across the substrate 3, as shownin FIG. 32( i), displacing the gas 72 to provide a layer 73 comprisingapolar medium extending across the openings of the compartments 4 incontact with the volumes 2 comprising polar medium, as shown in FIG. 32(j). As an alternative to being flowed, the layer 73 of apolar medium 74could be provided across the substrate 3 using some other technique suchas spraying.

In each of the first and second approaches, the displacement fluid flowsacross the support 3, through the gaps in the outer portions 23 andtherefore scrapes across the openings of the compartments 4 to displaceor clip the excess polar medium. Thus, the geometry and physicalproperties of the outer portions 23 and the inner recesses 22, includingthe effect of the indentations 65 when present, control the process ofdisposing the volumes 2 of polar medium in the inner recesses 22. Theeffectiveness of clipping and the ultimate shape of the volume 2 ofpolar medium is determined by a number of factors such as the relativeheights of the outer portions 23 and inner recesses 20, the aspect ratioof the inner recesses 20. The dimensions of the inner recesses 22 andthe outer portions 23 of the partitions 6 are ideally selected so thatthe volumes 2 comprising polar medium form a meniscus across the innerrecess 22 as shown in FIGS. 32( h) and (j).

By way of a counter-example, FIG. 33( a) shows steps of a methodcorresponding to that of FIG. 32, except that the support 3 having alarger ratio of pillar height to depth of the inner recess compared tothat of FIG. 32. Due to the increased pillar height, clipping of thepolar medium by the displacing fluid 74 takes place at the outer edge 15of the pillar as opposed to the outer edge 10 of the inner recess asshown in FIG. 33( e). This results in a larger volume of polar mediumbeing retained in the compartment 4 as shown by FIG. 33( f). Due to theincreased volume of the polar medium in the compartment a largerinterface is formed following flowing of the polar medium over thesupport, as shown in FIG. 33( h). In general the smaller the membraneinterface, the lower the noise and electrical resistance. Thus themembrane interface as shown in the method of FIG. 32 is preferred to themembrane interface as shown in the method of FIG. 33.

As regards specific dimensions of the geometry, it should be noted thatthe optimum dimensions are very much dependent upon the material system,including respective surface energies of the material of the substrate3, the apolar medium and the polar medium. Also because filling is adynamic process, it also depends to some extent upon the flow rateacross the substrate 3. Thus the preferred dimensions dependent on thematerial system. Any reference to particular dimensions herein hold fora material system where the substrate 3 is the epoxy resin TMMS, theapolar medium is silicone oil AR20 and the polar medium is 1M KCl.

As an alternative to flowing polar medium 71 across the support into thecompartments 4 and then removing the excess polar medium by flowing adisplacement fluid, the volumes 2 could be disposed on the support byinjecting discrete volumes 2 of polar medium into the compartments 4through air, for example using a printing technique. In that case theapolar medium 74 is then subsequently disposed on the support.

The pre-treatment apolar medium 70 also has a beneficial role in theformation of volumes 2 of polar medium.

Firstly, in the case of compartments 4 having gaps therebetween, thepre-treatment apolar medium 70 sits in the gaps and seals them againstflow of the polar medium. This assists in forming discrete volumes ofpolar medium by reducing the tendency of the volumes in neighbouringcompartments to contact and merge.

Secondly, the pre-treatment apolar medium 70 may also serve to coat thesupport 3 and may modify the surface properties in a beneficial way.Depending on the surface properties of the support 3 and the propertiesof the pre-treatment apolar medium 70, the addition of pre-treatmentapolar medium 70 may change the contact angle between the support 3 anda volume of polar medium disposed within a compartment. Thepre-treatment apolar medium 70 may be used for example to increase thephobicity of the support 3 to the polar medium and provide a volumehaving a more convex shape. The use of a pretreatment to alter thephobicity of the support 3 to a desired level permits the use of a widernumber of materials to be considered in making the support 3. This canbe useful for example in the case where a particular material isdesirable from a manufacturing point of view but does not haveappropriate material properties.

The aspect ratio of the inner recesses 22 is an important consideration.Aspect ratios (length:width) that are too large can result in a meniscusof the pre-treatment apolar medium 70 forming which spans the electrode13 as illustrated in FIG. 34( b). If the aspect ratio (depth d:width w)is too small, clipping can result in the removal of polar medium fromthe compartment. Desirably, the inner recesses 22 have a ratio of depthto width, where the width of an inner recesses is defined as thediameter of the largest notional sphere that can be accommodated withinthe inner recess 22, that is at least 1:3, preferably at least 2:3.Desirably, the inner recesses 22 have a ratio of depth to width, wherethe width of an inner recesses is defined as the diameter of the largestnotional sphere that can be accommodated within the inner recess, thatis at most 3:1, preferably at most 3:2.

The effectiveness of clipping and the ultimate shape of the volume ofpolar medium is determined by a number of factors such as the relativevalues of height (h) of the outer portions, the depth (d) of the innerrecess, the width (w) of the inner recess and the length (l) between arespective outer portion and an inner recess of a compartment, as shownin 34(a). The optimum dimensions for forming the array also depend uponfactors such as the relative surface energies of the material of thesupport, the apolar medium and the polar medium. The process for formingan array also depends upon the flow rate of the apolar and polar mediaacross the support.

A computer simulation of the second approach will now be described.

FIG. 35 shows the start point of the computer simulation, where thesubstrate 3 has been pre-treated with a pre-treatment apolar medium 70,in this example oil, and then filled with a polar medium 71, in thisexample an aqueous buffer solution. FIG. 35 also shows the front of theapolar medium 74 in its start point before being flowed across thesubstrate 3.

FIGS. 36(A) and (B) show the computer simulation after the apolar medium74 has flowed across the substrate 3, FIG. 36(A) showing the initialstate and FIG. 36(B) showing the steady state after the system has beenallowed to relax. FIG. 36(B) shows that the volume 2 of polar medium haspinned to the surfaces of the inner recess 22.

FIG. 36(C) shows a confocal image of a compartment 4 of the substratecontaining a volume 2 of polar medium. As predicted by the computersimulation, the volume 2 of polar medium has pinned to the surfaces ofthe inner recess 22.

FIG. 37 shows a relaxation from the initial to steady state, similar tothat of FIGS. 36(A) and (B), in simulations for inner recesses 22 havingwidths of 130 μm, 110 μm and 90 μm, all of which show pinning of thevolume 2 of polar medium to the surfaces of the inner recess 22. Theseresults also show that the meniscus of the volume 2 of polar medium, atits interface with the apolar medium, does not protrude above the edgesof the inner recess 22, which helps to achieve membrane size control.

FIGS. 38(A) and (B) are images showing the formation of volumes 2 ofpolar medium, in this case aqueous buffer solution, in the constructionof FIG. 21. Uniform volumes 2 of polar medium were observed, pinned tothe surfaces of the inner recess 22.

FIGS. 39(A) and (B) are images showing the formation of volumes 2 ofpolar medium, in this case aqueous buffer solution, in the constructionof FIG. 19. Uniform volumes 2 of polar medium were observed, pinned tothe surfaces of the inner recess 22.

The pre-treatment apolar medium 70 may comprise the amphipathicmolecules but this risks the amphipathic molecules providing anelectrically insulating layer across the electrode 13, so it ispreferred that the pre-treatment apolar medium 70 does not comprise theamphipathic molecules.

The layer 73 comprising apolar medium may comprise amphipathicmolecules. The apolar medium 74 which is flowed across the substrate 3may comprise the amphipathic molecules. Alternatively, the apolar medium74 which is flowed across the substrate 3 may not comprise theamphipathic molecules so that the initially provided a layer 73similarly does not comprise the amphipathic molecules, in which case theamphipathic molecules may be subsequently added to the layer 73comprising apolar medium.

In any of those cases, after formation of the layer 73 comprising apolarmedium, the apparatus 1 is left for a period of time that allows theamphipathic molecules to migrate to the interface between the layer 73comprising apolar medium and the volumes 2 comprising polar medium.Typically the apparatus 1 may be incubated for a period of time of theorder of 30 mins.

As another alternative, the amphipathic molecules may be provided in thepolar medium 75 that is subsequently flowed across the support 3 asdescribed below. A method of forming an array of membranes using theapparatus 1 is performed by forming an array of volumes 2 by the methoddescribed above, and then performing the following steps. These stepsare illustrated in FIG. 32 for that method of forming an array ofvolumes 2 of polar medium but is generally applicable to any of themethods of forming an array of volumes 2 of polar medium describedherein.

Polar medium 75 is flowed across the support 3 to cover the openings ofthe compartments, as shown in FIG. 32( k). The polar medium displacesthe apolar medium of the layer 73 comprising apolar medium to form alayer 76 comprising polar medium extending across the openings in thesupport 3 as shown in FIG. 32(1). FIG. 40(C) shows a more detailed view.The layer 75 comprising polar medium is brought in contact with thevolumes 2 comprising polar medium forming an interface 77 with each ofthe volumes 2 comprising polar medium.

In the case that the amphipathic molecules are already present membranes78 comprising amphipathic molecules are formed at the those interfaces77. This occurs simply by flowing the polar medium 75 over the support3.

Alternatively, the amphipathic molecules may be provided in the polarmedium 75 that is subsequently flowed across the support 3. In thatcase, after formation of the layer 75 comprising polar medium, theapparatus 1 is left for a period of time that allows the amphipathicmolecules to migrate to the interfaces 77 between the layer 75comprising polar medium and the volumes 2 comprising polar medium, andthereby form the membranes 78. Typically the apparatus 1 may beincubated for a period of time of the order of 30 mins. FIG. 31 shows anequivalent example for the case that the volume 2 of polar medium is adroplet in the apolar medium introduced into the compartment 4 using anemulsion as described above, showing the layer 50 of polar medium thathas been formed by flowing polar medium across the support 3 in the sameway.

The geometry and physical properties of the outer portions 23, includingthe effect of the indentations 67 when present, control the geometry ofthe layer 75 comprising polar medium extending across the support 3. Thedimensions of the inner recesses 22 and the outer portions 23 of thepartitions 6 are selected having regard to the dimensions of the innerrecesses 22 so that the volumes 2 comprising polar medium form ameniscus across the outer portions 23 as shown in FIG. 32(1). Themeniscuses of the volumes 2 comprising polar medium and the layer 75comprising polar medium extend towards each other to an extent thatbrings them into contact. Thus the geometry controls the formation ofthe membranes 78 providing reliability in that formation. This alsoallows control of the size and stability of the membranes 78 comprisingamphipathic molecules.

The relative heights of the pillars to the inner recesses is therefore adesign consideration. In the case of a particular material system wherethe substrate 2 is made of epoxy resin TMMS, the apolar medium issilicone oil AR20 and the polar medium is 1M KCl, when the height of thepillar was 60 μm and the height of the inner recess was 90 μm (1:1.5),clipping of the volume 2 of polar medium took place at the upper edge ofthe partition 6, which resulted in a volume 2 of polar medium whichprotruded from the inner recess. This resulted in a ‘muffin’ shapeddroplet with a large membrane area (large interface). Whilst thismembrane can work, it is not an ideal shape, as larger membranes areprone to more leaks, have a higher capacitance and are oftenelectrically more noisy. In the case of the material system mentionedabove, ratios of the height of the pillars to the inner recesses of30:90 and 30:120 were shown to be effective.

By way of example, FIGS. 40(A) and 40(B) are images of a support 3 withthe construction of FIG. 19 in which membranes have been formed in thecase of the polar medium being an aqueous buffer solution.

The apparatus 1 may be kept in the state with or without the layer 75 ofpolar medium, in storage or during transport from a manufacturingfacility to the point of use of the apparatus 1. The layer 50 of polarmedium may be applied after such storage or transport, if not alreadypresent.

In the first type of method of forming the volumes 2 of polar mediumwherein the volumes 2 of polar medium are pre-formed as droplets in anemulsion, in order for the droplets to be incorporated into thecompartments 4, they need to be provided within a fairly narrow range ofsize distribution and therefore it is necessary for the emulsion to bestable. The formation of a stable emulsion may be achieved by thepresence of amphiphilic molecules which form interfaces between thedroplets and apolar medium. In the absence of amphiphilic molecules, theemulsion is unstable. This tends to result in some degree of merging ofthe droplets to form larger droplets which are unable to fit correctlywithin the compartments 4.

A potential drawback however with the method of providing a stableemulsion is that during the process of filling the compartments 4, theapolar medium tends to coat the surfaces of the electrodes 13 providedin each compartment 4 resulting in a layer between the electrode 13 andthe volume 2 of polar medium that is an electrically resistive.Electrical contact between the electrode 13 and the volume 2 of polarmedium may be necessary requirement if it is desired to sense electricalsignals such as ion flow across a membrane. The presence of amphiphilicmolecules in the layer across the electrode 13 further exacerbates theproblem of poor electrical contact. Due to the presence of both apolarand polar groups, it is difficult to displace amphiphilic molecules fromthe surface of the electrode 13 by modifying its surfacecharacteristics.

The second type of method may be applied to reduce the problem of poorelectrical contact by assembling individual volumes 2 of polar medium inthe compartments 4 in the absence of amphiphilic molecules. The apolarmedium added to the substrate 3 is largely localised at the surface ofthe partitions 6 and away from the electrode 13. Thus, the pre-treatmentapolar medium 70 may further comprises the amphipathic molecules, sothat the membranes comprising amphipathic molecules are formed after thestep of flowing polar medium 8 across the support 3 to displace apolarmedium and form a layer of polar medium.

However, if the pre-treatment apolar medium 70 does not includeamphipathic molecules, the volumes 2 of polar medium are assembled inthe absence of the stabilising amphiphilic molecules, and so merging ofvolumes between neighbouring compartments is much more of an issue. Assuch, semi-closed structures are preferred (structures comprisingpartitions having no or few gaps provided on the surfaces of wells) dueto the fact that the individual volumes are confined within the wells.However the method will also work to some extent with open structures(pillars having gaps that extend the height of the compartments) due tothe fact that the pre-treatment apolar medium 70 can, depending upon theseparation between the pillars, partially span the gaps between thepartitions thus effectively providing a semi-closed structure.

The apparatus 1 may have membrane proteins inserted into the membranescomprising amphipathic molecules formed at the interfaces 51. Themembrane proteins may be ion channels or pores.

Such membrane proteins that are capable of insertion into the membranescomprising amphipathic molecules may initially be provided in either orboth of the layer 50 of polar medium and the volumes 2 of polar medium,prior to bringing the layer 50 of polar medium and the volumes 2 ofpolar medium into contact. In some material systems, bringing the layer50 of polar medium and the volumes 2 of polar medium into contact toform the membranes comprising amphipathic molecules may cause themembrane proteins to spontaneously insert into the membranes. Insertionof the membrane proteins into the membrane can be assisted wherenecessary for example by means such as the application of a potentialdifference across the membrane 2.

Alternatively the membrane proteins may be provided in the apolarmedium.

The membrane proteins may be used to perform analysis of a sample in thelayer 50 of polar medium.

To facilitate this, the layer 50 of polar medium may comprise the sampleto be analysed at the time it is initially added. As an alternative, thelayer 50 of polar medium may be provided as described above without thesample to be analysed. This allows the apparatus to be prepared forstorage and transportation prior to use. In that case, prior toperforming the analysis, there may be carried out a step of displacingthe layer 50 of polar medium by a further layer of polar medium thatcomprises the sample to be analysed.

Membrane proteins that are ion channels may be used to measure thetranslocation of an analyte through the ion channel by measurement ofcurrent flow under a potential difference applied across the ionchannel. The membrane itself is highly resistive and has a resistancetypically of the order of 1 GΩ or greater. Thus ion flow takes placessubstantially exclusively through the ion channel By way of example,FIG. 41 shows electrical data obtained for measurement of ion currentflow through an MspA nanopore illustrating pore insertion.

The ion channel may be a nanopore for determining the sequence of apolynucleotide. The current may be measured wherein the magnitude andduration of each current episode may be used to determine the sequence.The array may comprise an enzyme for control of translocation of thepolynucleotide through a nanopore.

The ion channel may be provided in the layer 50 of polar medium externalto the volumes 2 of polar medium. It is possible that more than one ionchannel may insert into the membrane or none at all. In practice therewill be a Poisson distribution of ion channels in the membranes.Following insertion of the ion channels, the membranes may be measured,for example by measurement of ion flow through the channel, in order todetermine which membranes contain a single ion channel. Dropletscontaining a single channel may be selected for further experimentation.The percentage of droplet interfaces containing single ion channels maybe optimised by varying the concentration of ion channels in the polarmedium.

Alternatively the ion channel may be provided in the apolar medium.Formation of an ion channel in a membrane may be checked optically byfor example providing a fluorophore in the polar interior of the dropletand a quencher in the polar meniscus layer. If an ion channel is presentin the membrane at the interface 51, the quencher and fluorophore willcome within close proximity of one another, extinguishing thefluorescent signal.

The magnitude of ion flow is dependent upon the potential differenceapplied across the ion channel and therefore is it desirable to providea stable reference potential. Both members of the redox couple arerequired in order to provide a stable reference potential. However, onemember may be provided and the other member generated in situ, forexample by oxidation or reduction of the redox member present.

Electrical measurements may be taken as follows.

The apparatus 1 further comprises a common electrode 60 arranged abovethe support 3 as shown in FIG. 31 so that the common electrode 60 makeselectrical contact with the layer 50 of polar medium once it has beenprovided.

As shown in FIG. 42, the apparatus 1 further comprises an electricalcircuit 61 connected between the common electrode 60 and the respectiveelectrodes 13 in each compartment 4. The electrical circuit 13 isarranged to take the electrical measurements and may have a conventionalconstruction, for example as discussed in more detail in WO-2009/077734which is incorporated herein by reference.

The electrical circuit 61 is configured to take electrical measurementsdependent on a process occurring at or through the membranes. Where asample containing an analyte is provided, for example in the layer of apolar medium, the process may analyse the sample. The polar medium ofthe layer 50 applied to the support 3 may be for example the liquidsample to be analysed. This sample may be a biological sample such asblood, serum, urine, interstitial fluid, tears or sperm. The liquidsample may be derived from a solid or semi-solid sample. The sample maybe agricultural, environmental or industrial in origin. It may be aforensic sample.

An electrochemical measurement apparatus typically comprises working,counter and reference electrodes wherein a potentiostat measures thepotential difference between the working and reference electrodes andmeasures current flow between the working and counter electrodes.Because no current flow takes place through the reference electrode aconstant potential difference is maintained between the referenceelectrode and working electrode. Alternatively, a two electrode systemmay be employed, as is the case with the apparatus 1, wherein apotential is provided between a counter and a counter/referenceelectrode and ion flow takes place between these electrodes. Thishowever results in consumption of one or the other member of the redoxcouple depending upon the polarity of the potential applied. The rate ofconsumption of the redox member is dependent upon the magnitude of theion flow.

In the case of measurement of the translocation of a polynucleotide, thepolynucleotide is caused to translocate the pore under a positivepotential applied across the pore. Application of a positive potentialresults in the oxidation of one member of the redox couple whichultimately will become depleted. Once depletion of a redox memberoccurs, the reference potential will start to drift, therefore limitingthe lifetime of the measurement. In the case of one or both members ofthe redox couple provided within the droplet the lifetime of themeasurement is dependent upon the amount of the reduced member of theredox couple, which in turn is dependent upon the concentration of theredox member and the droplet volume.

The apparatus 1 provides a stable array of volumes 2 of polar medium onwhich membranes may be formed in-situ. Such an array has advantages overan apparatus comprising an array of individual apertures across whichsuspended amphipathic membranes are provided. In the latter case, it ispossible that leakage can occur at the membrane edges over time. Bycontrast volumes 2 of polar medium contained in an apolar medium areextremely stable. Amphipathic membranes formed from triblock copolymersare very stable and resistant to biological degradation. However it hasproved very difficult to provide amphipathic membranes made fromtriblock copolymers, in particular silicon triblock copolymers, acrossan array of microwell apertures by methods such as described inWO2009/077734. By contrast it is relatively straightforward to preparesilicon triblock droplets. This enables nanopore arrays to be providedhaving very stable membranes and having a low susceptibility tobiological attack. This also enables the direct application of samplessuch as biological samples to the amphipathic membrane.

The apparatus 1 would typically be single use. Thereafter the componentsof the apparatus 1, namely the biological sample, the volumes 2 of polarmedium and apolar medium may be simply removed from the support 3, andthe support 3 cleaned and repopulated with volumes 2 of polar medium andapolar medium. This allows reuse of the silicon chip and the electrodearray comprising the array and the electrodes, which are expensivecomponents of the array chip. It also allows for replenishment of theredox couple.

A particular application is wherein the apparatus 1 is housed in asingle use handheld device for use with a computation means such as alaptop. Data is generated by the device and transmitted to thecomputation means by USB or other transmission means. The computationmeans would typically comprise a stored algorithm by which to generateevent and base calling data.

Alternatively the apparatus 1 could be housed in a reusable devicewherein the device comprises flow conduits allowing the array to becleaned by flushing with solution stored in on-board fluid reservoirs.

Having regard to the electrical requirements, the electrodes 13 may bearranged as follows.

The electrodes 13 provide an electrical contact to the volumes 2 ofpolar medium and may be used to provide a potential difference acrossthe membrane of amphipathic molecules. Electrical connections may extendfrom the electrodes 13 through the support 3 to an electrical circuit.

The electrodes 13 may be of any shape, for example circular. Anindividual electrode 13 may extend across the whole width of acompartment 4 or across a partial width thereof. In general, theelectrodes 13 may protrude above the base 5 or may be integral with thebase 5.

Some or all surfaces of the compartments 4 may be hydrophobic, includingthe outer surfaces of the partitions 6 inside the compartments 4. Thisassists positioning of a volume 2 of polar medium on an electrode 13 andthereby facilitates the making of an electrical contact.

The electrodes 13 may include other features to assist the making of anelectrical contact to the volumes 2 of polar medium.

One option is for the exposed surfaces of the electrodes 13 may beroughened, for example by provision of a layer of Pt black on a Ptelectrode.

Another option is for the electrodes 13 to comprise spikes 16 protrudinginto the compartment 4 to penetrate the volumes of polar medium.Following penetration of a volume 2 of polar medium by a spike 16 ittends to reform around the electrode effectively resealing the volume 2of polar medium.

Exposed surfaces of the electrodes 13 may be hydrophilic and/or surfacesof the compartments 4 around the electrodes 13, for example the exposedsurfaces of the surface coating 14, that may be hydrophobic. This canreduce the tendency of the apolar medium to coat the exposed surfaces ofthe electrodes and thereby act as an electrically insulating layer.

The electrode 13 may be a reference electrode such as Ag/AgCl in orderto provide a stable reference potential with respect to a counterelectrode. Alternatively the electrode 13 may be an electrochemicallyinert material such as Au, Pt, Pd or C and the electrode potentialprovided by one or both members of a redox couple located within thepolar interior of the droplet. Types of redox couples that may beemployed are for example Fe(II)/Fe(III), Ru (III)/Ru (II) andferrocene/ferrocinium. Specific examples of redox couples that may beemployed are ferri/ferrocyanide, ruthenium hexamine and ferrocenemonocarboxylic acid.

FIG. 43 shows the droplet lifetime for various droplet diameters forferro/ferricyanide as the redox couple. As can be seen from the graph, a200 mM concentration of ferrocyanide in a 200 μm diameter droplet has alifetime of approximately 140 hrs for a current flow of 100 pA.

The support 3 is designed as follows to assist the formation ofmembranes of amphipathic molecules.

As shown in FIG. 31, the layer 50 of polar medium forms a meniscus 57that protrudes into the compartments 4 to contact the volumes 2 of polarmedium. All the constructions of the support 3 described above providethe advantage that the upper surfaces of the partitions 6 assist in theformation and pinning of the meniscus 52. In particular, the various,convoluted shapes of the upper surface of the partitions 6 providespinning points for the layer 50 of polar medium in order to form themeniscus 52.

A meniscus formed in a conventional square type of well structure is notpinned uniformly around the edges of the well. As such, stresses on themeniscus are created at the corners of the well. In order to optimisemeniscus formation in a well type structures, it is beneficial toprovide further features on the wells in order to pin the polar layer.All the constructions of the support 3 described above provide suchfeatures, being for example the convoluted shapes of the various pillars7 and 23 and the undulating shape of the common body 31 around therecess 30. The meniscus 52 may be pinned around these undulationseffectively creating a more distributed pinned meniscus 52 in thecompartment 4.

In general terms, such pinning is achieved in the constructions of thesupport 3 described above because in each case the total length percompartment 4 of the edges of the outer ends of the partitions 6 in thecommon plane is greater than the largest circumference of the largestnotional sphere that can be accommodated within the compartments 4.

The layer 50 of polar medium forms the meniscus 52 with the partitions 6which extends into the compartment 4 and contacts the volume 2 of polarmedium provided therein to form a membrane. The compartments 4 aredesigned with openings having dimensions selected so that the layer of apolar medium when applied will form a meniscus 52 extending into thecompartment 4 to an extent that brings the layer 50 of polar medium intocontact with at least some of the volumes 2 of polar medium.

The ability to form a membrane is dependent upon the height of thevolume 2 of polar medium within the compartment 4 and the extent towhich the meniscus 52 extends into the compartment 4. This in turn isdependent upon the surface interaction between the partitions 6, thepolar medium and the apolar medium, as well as the dimensions and shapeof the compartment 4 defined by the partitions 6. These parameters,and/or the sizes of the volumes 2 of polar medium, may be selected suchthat a polar medium applied to the top surface of the support 3 willspontaneously form membranes with the volumes 2 of polar medium.

This is illustrated schematically in FIGS. 44( a) to (c) for the casethat the volumes 2 of polar medium are droplets in the apolar medium.FIG. 44( a) shows the case that there is no contact between the layer 50of polar medium and the volume 2 of polar medium so that a membrane isnot formed due the volume 2 of polar medium being too small and/or themeniscus 52 not extending sufficiently into the compartment 4.

FIG. 44( b) shows the case whereby the volume 2 of polar medium and themeniscus 52 just contact one another. However, the size of the membranemay be insufficient. Also the membrane formation may be sensitive toother parameters. The size of the volume 2 of polar medium istemperature dependent and a small drop in temperature can result incontraction of the volume 2 of polar medium leading to the non-formationof a membrane. Furthermore, whilst the volumes 2 of polar medium aredesigned to be substantially similar in size, a small variation in thedroplet size may occur, resulting in unreliable membrane formation.

FIG. 44( c) shows the case where the volume 2 of polar medium is madelarger and/or the meniscus 52 extends further into the compartment 4such that a substantial droplet interface is formed. This reduces thechances that the membrane will not be formed as well as providing alarge surface area for ion channel insertion.

FIGS. 45 and 46 are schematic cross-sectional views of the apparatus 1of the type shown in FIGS. 15 to 18 wherein the partitions 6 compriseinner portions 20 and outer portions 21 that comprise pillars 23 havinggaps 24 therebetween, for the case that the volumes 2 of polar mediumare droplets in the apolar medium. FIGS. 45 and 46 show the influence ofthe height and density of the pillars 23 on the pinning of the meniscus52.

In FIG. 45, the meniscus 52 is pinned at the edges of the inner portions20 and not the pillars 23. Additional apolar medium is pinned at theinterface between the pillar 23 and the edge of the recesses 22 of theinner portion 20. The pillars 23 therefore serve to control thedistribution of apolar medium but do not influence the formation of themeniscus 52 which is controlled by the recesses 22.

In FIG. 46, the arrangement of the pillars 23 is such that the meniscus52 is determined by the pillars 23 themselves and not the recesses 22 inthe inner portions 20. In FIG. 46, two sets of pillars 23 are providedbetween neighbouring compartments 4 on the inner portions 23 of thepartitions 4. Alternatively for example, a single pillar might beprovided having a larger height than that shown in FIG. 1.

Whether the meniscus 52 forms according to that of FIG. 45 or 46 willdepend upon the arrangement and relative dimensions of the pillars 23 ofthe outer portions 21 and the recesses 22 of the inner portions 20.

Compartments 4 with no gaps between the partitions 6 have the tendencyto flood with apolar medium. This is disadvantageous as this preventsformation of the membrane interface between the two volumes 2 of thehydrophilic medium.

FIG. 47 shows a meniscus 52 formed by a polar liquid at the surface ofthe support 3. The size and degree of curvature of the meniscus 52 ofthe layer 50 of polar medium applied to the upper surface of the support3 can be controlled across a wide range. The curvature of the meniscus52 will be determined by the contact angle between the polar liquid andthe partitions 6, which is a property of the material system of thepolar medium, the apolar medium and the surface properties of thepartitions 6. It will also be determined by the dimensions across theopening of the compartment 4 on which the meniscus 52 is formed and theheight of the partitions 6.

For example, for a compartment 4 having a width b between the partitions6, a height c and a contact angle θ between the surface of the pillarand the meniscus 52, a meniscus 52 will be formed above the base of thecompartment when:

$\frac{c}{b} \leq \frac{1 - {\sin \; \theta}}{2 - {\cos \; \theta}}$

The distance h that the meniscus 52 extends into the compartment can bedetermined, and can be controlled in combination with the size of thevolumes 2 of polar medium to control the size of the meniscus 52.Conceptually, assuming a perfectly spherical volume 2 of polar medium ofdiameter d, an interface will be formed between the meniscus 52 and thevolume 2 of polar medium when the diameter d≧c−h. For a diameter d of150 μm, Permex being the material of the partitions 6 and a the polarmedium of the layer 50 being 1M HEPES, suitable values for b and c areb=150 μm, a=30 μm, c<170 μm.

For a pillar array, menisci 52 are formed on the partitions 6 in what isknown as a superhydrophobic or Fakir state. The Fakir state occurs when:

${\cos \; \theta} < \left( {{- 1} + {4\frac{c}{a}\varphi_{s}}} \right)$

where a is the pillar thickness and where Φ_(s) is a dimensionless termwhich is equal to the fraction of solid in contact with the liquid.

Although the above examples are given assuming a perfectly sphericalvolume 2 of polar medium for ease of understanding, this might not bethe case. In the case of a gel, the volume 2 of polar medium may bedesigned to have other shapes. Furthermore, even if the shape prior toentering the compartment 4 is spherical in shape, it may deform to someextent depending upon the nature of interaction with the support 3and/or surface of the electrode 13, thus changing the height of thevolume 2 of polar medium after it is contained in the compartment 4.This factor also needs to be taken into account when assessing theheight of the volume 2 of polar medium in order to be able tospontaneously form membranes.

The widths and heights of the compartments 4 may be selected as follows.To take account of the differing profiles of the compartments 4 acrossthe support 3, for this purpose, the width of a compartment 4 is definedas the diameter of the largest notional sphere that can be accommodatedwithin the compartment 4.

The width of the compartment 4 may be chosen to be a value less than 2times the average diameter of the volumes of polar medium in order toavoid the possibility that more than one volume 2 of polar medium may becontained in a side by side relationship within a compartment 4 wheredesirable. Where the volume of polar medium is a liquid droplet, thecompartment width may have a value less than 2 times, for example 1.75times the width, to take account of the fact that the droplets maydeform, thus reducing their average width.

For the case that the volumes 2 of polar medium are droplets in theapolar medium, the width of the compartment 4 may further be chosen tobe a value greater than the average diameter of a volume 2 of polarmedium such that it may freely insert into the compartment 4. For thispurpose, the width will typically be at least 1.05 times the averagediameter of the volumes 2 of polar medium. The width will typically beat most 1.5 times the average diameter of the volumes 2 of polar medium.Widths greater than this provide the possibility that the volume 2 ofpolar medium may move within the compartment 4. Ideally the volume 2 ofpolar medium is provided in a closely packed arrangement within thecompartment 4.

The height of the compartment 4 is determined by the height of thepartitions 6. The height of the compartment 4 is chosen depending uponthe size of the volumes 2 of polar medium and the ability to form amembrane with a polar liquid. The pillar height is typically between 1.1and 1.3× the height of the droplet in the droplet zone. The compartments4 may have heights that are at least 1.1 times the average diameter ofthe volumes of polar medium. The compartments 4 may have heights thatare at most 1.3 times the average diameter of the volumes of polarmedium. In a particular embodiment where the volume of polar medium is abead, it may extend beyond the height of the partitions.

Some examples of experiments performed using an apparatus 1 as describedabove will now be given. In various examples, the apparatus 1 was formedas an array chip.

The first example is as follows.

Droplets of polar medium in apolar medium may be prepared as follows. 2mg/ml of 6-30-6 PMOXA-PDMS-PMOXA triblock copolymer was dissolved inAR20 silicon oil to provide the apolar medium. The polar mediumconsisting of 625 mM NaCl, 75 mM potassium ferrocyanide and 25 mMpotassium ferricyanide in 100 mM HEPES. Droplets were prepared in amicrofluidic T-junction (Chip type: Dolomite, part no. 3000158) havingtwo intersecting channels of 300 μm width. The channels narrow towardsthe intersection to provide channel widths at the intersection of 105μm. The polar and apolar solutions were flowed along the channels atrespective solution flow rates of 3-4 ul/min and 15-17 ul/min to providedroplets having a droplet size of between 150-160 μm.

The flow rates can be varied to provide droplets of differentdimensions.

A silicon wafer having an array of Pt connectors spaced apart by 200 μmby 225 μm was coated with a 2-3 μm layer of SU photoresist. Permexpillars were added to the base support by a standard photolithographicprocess wherein uncrosslinked Permex precursor is applied to the baseand the precursor cross linked by exposure to UV light through apatterned mask. The uncrosslinked precursor was subsequently washed awayto reveal the pillar structure. The resulting array was a 38×128 dropletzone with a pillar shape according to FIG. 2. The pillar height was 160μm. Droplets of 145 μm in diameter were added to the array in the formof an AR20 oil/droplet emulsion as described above.

The emulsion was applied to the top surface of the array and oil anddroplets were drawn into the array by capillary action. Excess dropletswere removed by flowing AR20 silicon oil over the array. The dropletswere held in the array by surface tension.

The array was placed into a flow cell and the polar medium containing anMspA protein nanopore was flowed over the surface of the array toprovide ion channels in the droplet interfaces.

Droplet interfaces containing single MspA nanopores were selected forexperimentation and measurement of DNA was carried out by measuring ionflow through the individual nanopores during translocation of DNA.

The second example is as follows.

Apparatuses were prepared according to the following general method.

Array chips were fabricated in clean-room facilities. A 6 in Si waferwith a 1 μm thermal oxide (SiMat) was used a base support. The wafer wasinitially treated with 200 W, O2 plasma in a plasma processor (OxfordInstruments), it then underwent a dehydration bake at 150° C. for 15minutes in a hotplate (Electronic Micro Systems Ltd). The wafer wascoated with a 1 μm layer of SU-8 2 photoresist (MicroChem Corp.) in aspin-coater (Electronic Micro Systems Ltd, 3000 rpm for 30 seconds),soft baked at 65° C. and 2 min at 95° C. in a hotplate, flood exposed(110 mJ/cm2) in a UV mask aligner (Quintel Corp.) and then post-exposurebaked (PEB) for 1 min at 65° C. followed by 2 min at 95° C. in ahotplate. The wafer is then spin-coated with a 120 μm thick layer ofSU-8 3050 (1250 rpm for 30 s) and soft baked for 5 min at 65° C.followed by 2.5 h at 95° C. in a level hot plate. After cooling, it isexposed (260 mJ/cm2) in the mask aligner using the photomask patternedwith the microfluidic network. A PEB is then carried out: 2 min at 65°C. and 15 min at 95° C. The channel features are developed by immersionof the wafer in Microposit EC Solvent (Rhom Haas Electronic Materials),in an appropriately size beaker, and shaken for 10 min and finallyrinsed. Once the channels have been formed, the wafer is treated with O2plasma for 1 min at 200W to promote adhesion of the top layer and theSU-8 resist.

The channels are sealed with a layer of 100 μm thick film laminateresist, SUEX (DJ DevCorp) using a Exclam-Plus laminator (GMP) with thetop roller set at 45° C., with a pressure setting at 1 mm thickness anda speed of 50 cm/min. A post lamination bake of 3 min at 65° C. was thencarried out. After cooling, the SUEX layer was exposed (1400 mJ/cm2)using the fluidic port mask. It should be noted that the protectivepolymer layer on the laminate is left on. The PEB is 3 min at 65° C.followed by 7 min at 95° C., again with the protective film on. Thewafer is then developed using propylene glycol monomethyl ether acetate(Sigma-Aldrich) for 10 min and rinsed thoroughly with isopropanol,making sure the all residual developer is rinsed from the interior ofthe channels. The wafers are finally hard baked at 150° C. for 1 h anddiced into individual array chips.

EXAMPLE 1

This example describes the method used to produce the triblockco-polymer droplets which were used to fill the interconnecting dropletzones on the array.

Materials and Methods

The T-junction chips were prepared for droplet generation by affixingnanoport assemblies (Upchurch Scientific) as fluidic interfaces.

The droplet generation mechanism in a T-junction is well documented inthe literature [Garstecki et al., Lab Chip, 2006, 6, 437-446 and Thorsenet al., Physical Review Letters, 2001, 86, 18, 4163-4166]. Taking intoaccount the fluid viscosities of the reagents involved the chosenT-junction geometry was 50 μm channel width for both cases (oil andbuffer).

1.1—Droplet Reagents

In order to make aqueous phase droplets in oil, buffer was used as thedisperse phase, while a silicon oil (e.g. AR20), was used as thecontinuous phase. Both buffer and triblock co-polymer-containing oilwere prepared as described below.

A solution of buffer (buffer 1) was prepared by adding 298 mg of KCl(99.99% Purity, Sigma) to 10 mL of degassed DI water. To this solution30.35 mg of 2-Amino-2-(hydroxymethyl)-1,3-propanediol (99.9%, Sigma) wasadded. The solution was buffered to pH 8 using small quantities of HCland NaOH. 316.5 mg of K2[Fe(CN)6] (99.9%, Sigma) and 82.3 mg ofK3[Fe(CN)6] (99.9%, Sigma) was added to the solution and stirred untildissolved.

Oil-triblock co-polymer solution was prepared by adding 20 mg of polymer(6-33-6, PMOXA-PDMS-PMOXA, PolymerSource) to 1 mL of AR20 (99%, Sigma).The polymer was left stirring in the oil for 24 hrs until all of thepolymer had dissolved.

1.2—Droplet Generation Setup

The droplet generation setup consisted of two syringe pumps (Elite,Harvard Apparatus), two gastight syringes (Hamilton), peak tubing(Upchurch Scientific), and a custom made T-junction microfluidic chip.Once the syringes were loaded with oil and buffer and mounted on thesyringe pumps, the peak tubing was used to establish the fluidicconnections to the ports on the chip. The oil syringe was connected tothe continuous phase channel input while the buffer was connected to thedisperse phase channel input.

Both syringe pumps were set to infuse at a flow rate of 10 μL/min, whichproduced an average droplet size (diameter) of 129.46 μm, with astandard deviation of 10.87 μm. The droplets were then collected in avial.

EXAMPLE 2

This example describes the method used to producedroplet-interface-bilayers (DIBs) using a number of different tri-blockco-polymers in different oils. The ability to form bilayers and to allowinsertion of biological nanopores (such as mutants of MspA) was alsoinvestigated.

Materials and Methods

Experiments 2.1, 2.3 and 2.4 were carried out on the below combinationsof tri-block co-polymer and oil.

1—6-33-6 (PMOXA-PDMS-PMOXA) PolymerSource (20 mg/mL) in AR20 oil(polyphenyl-methylsiloxane, Sigma Aldrich).

2—6-33-6 (PMOXA-PDMS-PMOXA) PolymerSource (20 mg/mL) in PDMS-OH 65cStoil (poly(dimethylsiloxane), hydroxyl terminated, Sigma Aldrich).

3—6-45PE-6 (PMOXA-PE-PMOXA, where PE=a polyelethylene hydrocarbon chainapproximately 45 carbon atoms in length.) PolymerSource (20 mg/mL) inhexadecane (99.9%, Sigma Aldrich).

4—6-32-6 (PMOXA-PDMS-PMOXA) HighForce (20 mg/mL) in AR20 oil(polyphenyl-methylsiloxane, Sigma Aldrich).

2.1—Droplet Stability Experiments

Droplet stability was measured off-line by preparing solutions of bufferand triblock ABA polymer in various oils. A small 0.5 cm² tray wasprepared using polycarbonate and a glass slide. The tray was filled withoil. To the oil, 1 μL buffer droplets were added and monitored over 24hrs. Droplets that exhibited only a small degree of merging wereprogressed to electrical DIBs testing.

2.2—Experimental Set-Up

The experimental system was as follows. A 700B axopatch was connectedinside a shielded box containing two micro-manipulators. The entirefaraday cage was placed on an inverted microscope (Nikon) such that itwas possible to view the manipulation of the droplets from underneath.This allowed the droplets to be moved without opening the Faraday cage.

Within the Faraday cage, the electrodes of the 700B axopatch wereconnected via pure gold (Au) wire

The Au was prepared for use in the droplet setup by flaming the end suchthat the wire formed a small gold bead. The Au wire was cleaned byemersion in conc.HNO₃ for 30 s, and washed thoroughly with DI water. Theball-ended wire was then repeatedly moved through a liquid agarosesolution prepared from the buffer (5% wt low-melt agarose, Lonza/Buffer400 mM KCl, 75 mM K2[Fe(CN)6] (99.9%, Sigma) and 25 mM K3[Fe(CN)6](99.9%, Sigma), 10 mM Tris). Once a small bead had formed on the end theagarose was allowed to cool, and the wire was stored in an excess ofbuffer solution in order to come to equilibrium.

The droplet chamber was mounted on the stage within the Faraday cage,and the electrodes were mounted such that both fell within the centralsection of the chamber. The manipulators were situated such that a fullrange of movement in X and Y directions were achievable by bothelectrodes over the area of the chamber. The chamber was then filled tothe brim with the AR20 tri-block co-polymer solution and allowed tostand for a few minutes. 1 μL of buffer was pipetted directly onto eachof the agarose tipped Au wires and both electrodes were moved directlyunder the AR20/triblock co-polymer solution. The droplets were leftunder the solution for 30 s before movement.

2.3—Bilayer Formation

To form a membrane with the droplet pair, a waveform of ±20 mV wasapplied to the electrodes in addition to a bias voltage of 180 mV. Thecurrent response was monitored as the indicator of the formation of acapacitive membrane. The droplets were carefully brought together suchthat contact between the two buffer volumes was made. The droplets wereleft in this state until a membrane was formed. In situations where themembrane growth was very slow, the droplets were moved in the XYdirection, which forced exclusion of the AR20/triblock co-polymerbetween the droplets and facilitated membrane growth.

2.4—Nanopore Insertion Experiments

In order to insert trans-membrane pores across the membrane, a 0.0005mg/ml solution of MspA-(B2C) (SEQ ID NO: 1 and 9) was added to thebuffer that formed the analyte. Insertion of the pore was observed by aninstantaneous increase in current. This was performed in the absence ofthe waveform, but under the applied bias potential.

Results

The different tri-block co-polymer and oil combinations that wereinvestigated are shown in Table 1 below.

TABLE 1 Off-line MspA- Tri-Block Stability Membrane (B2C) PoreCo-Polymer Oil Test Formation Insertion 6-33-6 AR20 stable capacitivepores PolymerSource droplets membrane inserted formed growth observed6-33-6 PDMS-OH stable capacitive pores PolymerSource 65cSt dropletsmembrane inserted formed growth observed 6-45PE-6 C16 stable capacitivepores PolymerSource droplets membrane inserted formed growth observed6-32-6 AR20 stable capacitive pores HighForce droplets membrane insertedformed growth observed

Capacitive membrane growth and pore insertion was observed for all ofthe tri-block co-polymer/oils tested. Membrane growth and MspA-(B2C)(SEQ ID NO: 1 and 9) pore insertion were observed for the 6-33-6PolymerSource tri-block co-polymer used with AR20 silicone oil. Membranegrowth and pore insertion were observed for the 6-45PE-6 PolymerSourceused with hexadecane as an example of a triblock co-polymer which doesnot have the PDMS central core structure.

EXAMPLE 3

This example describes the method used to produce the array chips whichare assembled with patterned interconnecting droplet zones.

Materials and Methods 3.1—Array Chip Formation 3.1.1 Array ChipFabrication

The array chips were fabricated in clean-room facilities. A 6 in Siwafer with a 1 μm thermal oxide (SiMat) was used as a base for thesupport. The wafer was initially treated with 200 W, O₂ plasma in aplasma processor (Oxford Instruments), it then underwent a dehydrationbake at 150° C. for 15 minutes in a hotplate (Electronic Micro SystemsLtd). The wafer was coated with a 1 μm layer of SU-8 2 photoresist(MicroChem Corp.) in a spin-coater (Electronic Micro Systems Ltd, 3000rpm for 30 seconds), soft baked at 65° C. and 2 min at 95° C. in ahotplate, flood exposed (110 mJ/cm²) in a UV mask aligner (QuintelCorp.) and then post-exposure baked (PEB) for 1 min at 65° C. followedby 2 min at 95° C. in a hotplate. The wafer was then spin-coated with a120 μm thick layer of SU-8 3050 (1250 rpm for 30 s) and soft baked for 5min at 65° C. followed by 2.5 h at 95° C. in a level hot plate.

After cooling, it was exposed (260 mJ/cm²) in the mask aligner using thephotomask patterned with the microfluidic network. A PEB was thencarried out: 2 min at 65° C. and 15 min at 95° C. The channel featureswere developed by immersion of the wafer in Microposit EC Solvent (RhomHaas Electronic Materials), in an appropriately size beaker, and shakenfor 10 min and finally rinsed. Once the channels had been formed, thewafer was treated with O₂ plasma for 1 min at 200W to promote adhesionof the top layer and the SU-8 resist.

The channels were sealed with a layer of 100 μm thick film laminateresist, SUEX (DJ DevCorp) using a Exclam-Plus laminator (GMP) with thetop roller set at 45° C., with a pressure setting at 1 mm thickness anda speed of 50 cm/min. A post lamination bake of 3 min at 65° C. was thencarried out. After cooling, the SUEX layer was exposed (1400 mJ/cm²)using the fluidic port mask. It should be noted that the protectivepolymer layer on the laminate was left on. The PEB was 3 min at 65° C.followed by 7 min at 95° C., again with the protective film on. Thewafer was then developed using propylene glycol monomethyl ether acetate(Sigma-Aldrich) for 10 min and rinsed thoroughly with isopropanol,making sure the all residual developer was rinsed from the interior ofthe channels. The wafers were finally hard baked at 150° C. for 1 h anddiced into individual chips.

3.1.2 Open Structure Array Chip Fabrication

The functional structure array chips were fabricated in clean-roomfacilities. A 6 inch Si wafer (Silex) containing bias and electrodes wasused as substrate. The wafer was initially treated with 200 W, O₂ plasmain a plasma processor (Oxford Instruments), it then underwent adehydration bake at 150° C. for 15 minutes in a hotplate (ElectronicMicro Systems Ltd). The wafer was coated with a 1 μm layer of SU-8 2photoresist (MicroChem Corp.) in a spin-coater (Electronic Micro SystemsLtd, 3000 rpm for 30 seconds), soft baked at 65° C. and 2 min at 95° C.in a hotplate, exposed (110 mJ/cm²) in a UV mask aligner (Quintel Corp.)with a Seed layer mask. The wafer was then post-exposure baked (PEB) for1 min at 65° C. followed by 2 min at 95° C. in a hotplate and developedin EC Solvent for 1 min. The Seed layer function was to improve adhesionof the high aspect ratio and it also ensured that only the desired areaof the electrodes was exposed to solution.

A layer of dry-film resist TMMF 2030 (Tokyo Ohka Kogyo Co. Ltd.) wasapplied to the wafer using an Excelam-Plus roll laminator (GMP Co. Ltd.)with a top roll temperature of 85° C. The process was then repeated fivetimes, to achieve a 150 μm thickness. The wafer was then exposed to UVin the mask aligner using a Pillar structure mask. A PEB at 95° C. wascarried out in a hotplate for 10 min previous to development of theresist in EC Solvent for 12 min. The wafer was then treated with a 200 WO₂ plasma and hard baked in an oven at 200° C. for 1 h.

At this stage the wafer with the formed pillar structure was diced intoindividual devices and packaged onto ASIC-containing PCBs.

3.1.3 Closed Structure Array Chip Fabrication

This type of functional structure was fabricated in the same basesubstrate as the open structure array chip fabrication, i.e. a Silexwafer containing bias and electrodes. A Seed layer was formed in thesame way as described in the previous section. Then the wafer waslaminated with four layers of TMMF 2030 dry-film resist, with the sameprocess parameters as described above. These four layers, with anoverall thickness of 120 μm, were then exposed using the Wells mask. Thewafer was subsequently laminated with a fifth layer of TMMF 2030 andexposed using the Pillars mask. The wafers then underwent a PEB at 95°C. for 10 min, were developed in EC Solvent for 12 min, 2 min O₂ plasmaat 200 W and a hard bake at 200° C. for 1 h.

At this stage the wafer with the formed well and pillar structures, wasdiced into individual devices and packaged onto ASIC-containing PCBs.

EXAMPLE 4

This example describes the method used to populate the array chips,which were assembled with patterned interconnecting droplet zones, withtri-block copolymer droplets formed using the method detailed in Example1.

Materials and Methods 4.1—Membrane Formation on Open Structure Arrays

To dispense the droplets onto the array of interconnecting dropletzones, a 1000 μL micro-pipette (Gibson) was used. The pipette tip wascut by 1 mm to, enlarge the orifice and prevent droplet merging due toshear stress. The droplets were then slowly dispensed onto the surfaceof the interconnecting droplet zones, ensuring that the entire area wascover with a large excess. Most of the excess droplet solution was thenremoved by inclining the array, in order to allow gravity to remove theexcess droplets which had not been captured in the droplet zones. Atthis point, a flow cell large enough to fit the entire area of the arraywas placed on top of it, sealed and then filled with oil. This step wascarried out because droplets can stick to one another and flushing theflow cell with oil removes the remaining droplets from the top of thecapture array. Finally, tri-block co-polymer membranes were formedbetween each individual droplet and a common aqueous volume by flushingthe bulk of the oil away and substituting it for an aqueous phase i.e.buffer 1. As the oil was displaced from the flow cell, the aqueoussolution came into contact with the top part of the capture structure aswell as the top of each droplet. The self assembled triblock copolymerlayer prevented the two aqueous phases from merging; providing thedroplet was big enough to be in contact with the bulk aqueous solution.The cross-section of the apparatus 1 is as shown in FIG. 31.

EXAMPLE 5

This example describes the insertion of MspA-(B2C) (SEQ ID NO: 1 and 9)pores into tri-block co-polymer droplets (6-33-6 PolymerSource dropletsin AR20 (Sigma Aldrich) and helicase controlled DNA movement through thenanopore. The droplets used in these experiments were made ofcross-linked agarose beads (Bio-Works) (140-150 μm) which had beencoated in tri-block co-polymer in AR20 silicone oil.

Materials and Methods 5.1—Agarose Bead Preparation

The cross-linked agarose beads were obtained from Bio-Works in a broadrange of sizes (130-250 μm). The droplets were then sieved using filtersto obtain beads that were in the size range 140-150 μm and stored inpure water. The beads were then centrifuged and buffer exchanged (625 mMKCl, 75 mM potassium ferrocyanide, 25 mM potassium ferricyanide, 100 mMCAPS, pH 10.0) at least 5 times. Immediately after the final bufferexchange and centrifuge step (to remove excess water) the beads wereextracted and immersed in 10 mg/mL 6-33-6 PolymerSource triblockco-polymer in AR20 silicone oil. The beads were briefly vortexed for30sec in the oil, and left to stand for 1 hour.

5.2—Membrane Formation

Cross-linked agarose beads in 6-33-6 PolymerSource triblockco-polymer/AR20 were added to the array, and manually inserted into theinterconnecting droplet zones. Immediately after filling, a small amountof 10 mg/mL 6-33-6 PolymerSource triblock co-polymer/AR20 (˜50 uL) wasadded to the surface of the array to immerse the beads and keep themunder oil. They were incubated in this state for 5 mins. After this thechip was assembled and buffer (625 mM KCl, 75 mM potassium ferrocyanide,25 mM potassium ferricyanide, 100 mM CAPS, pH 10.0) was immediatelyflowed through. The array was then ready for testing.

5.3—Pore Insertion and Helicase Controlled DNA Movement

In order for pores to insert into the triblock co-polymer, a solution ofbuffer (625 mM KCl, 75 mM potassium ferrocyanide, 25 mM potassiumferricyanide, 100 mM CAPS, pH 10.0) with MspA-(B2C) (SEQ ID NO: 1 and 9)was flowed over the array. A holding potential of +180 mV was appliedand pores were allowed to enter bilayers until at least 10% occupancywas achieved. Once pores had inserted, then buffer solution (625 mM KCl,75 mM potassium ferrocyanide, 25 mM potassium ferricyanide, 100 mM CAPS,pH 10.0) containing no MspA-(B2C) (SEQ ID NO: 1 and 9) was then flowedover the array to prevent further pores inserting into the tri-blockco-polymer. In order to observe helicase-controlled DNA movement, asolution containing DNA (SEQ ID NO: 3 connected via 4 spacer groups toSEQ ID NO: 4, 1 nM), helicase enzyme (100 nM), dTTP (5 mM), Mg2+ (10 mM)in buffer (625 mM KCl, 75 mM potassium ferrocyanide, 25 mM potassiumferricyanide, 100 mM CAPS, pH 10.0) was flowed over the array. A holdingpotential of +180 mV was applied and helicase-controlled DNA movementwas observed.

Results

Upon the exposure of the tri-block co-polymer covered agarose dropletsto MspA-(B2C) nanopores, insertion of the pores into the tri-blockco-polymer were observed. On the addition of DNA (SEQ ID NO: 3 connectedvia 4 spacer groups to SEQ ID NO: 4, 1 nM) and helicase enzyme to thesystem, helicase controlled DNA translocation through the MspA-(B2C)nanopore was observed. Two example current traces showinghelicase-controlled DNA movement through nanopores inserted into agarosedroplets are shown in FIGS. 48A and B.

EXAMPLE 6

This example describes the insertion ofalpha-hemolysin-(E111N/K147N)₇(SEQ ID NO: 5 and 6) pores into tri-blockco-polymer droplets (6-33-6 PolymerSource droplets in AR20 (SigmaAldrich) and how this system was used to detect the presence of theprotein thrombin. The droplets used in these experiments were made oflow melt agarose.

Materials and Methods 6.1—Agarose Bead Preparation

The droplet generation setup consisted of two syringe pumps (Elite,Harvard Apparatus), two gastight syringes (Hamilton), peak tubing(Upchurch Scientific), and a custom made T-junction microfluidic chip.Once the syringes were loaded with 6-33-6 triblock copolymer in AR20 oilin one and 2% low melt agarose (Lonza) in buffer (625 mM KCl, 75 mMpotassium ferrocyanide, 25 mM potassium ferricyanide, 100 mM CAPS, pH10.0) in the other and mounted on the syringe pumps, the peak tubing wasused to establish the fluidic connections to the ports on the chip. Theoil syringe was connected to the continuous phase channel input whilethe buffer was connected to the disperse phase channel input. In orderto make agarose droplets, the set-up was placed in an oven at 50° C. inorder for the agarose solution to remain fluid during the dropletgeneration process.

Both syringe pumps were set to infuse at a flow rate of 10 μL/min forthe agarose in buffer and 25 μL/min for the 6-33-6 in AR20 oil, whichproduced an average droplet size (diameter) of 150 μm, with a standarddeviation of 5 μm. The droplets were then collected in a vial.

6.2—Membrane Formation

The tri-block co-polymer membrane was formed as described in Example 5.

6.3—Pore Insertion and Detection of the Protein Thrombin

In order for pores to insert into the triblock co-polymer, a solution ofbuffer (625 mM KCl, 75 mM potassium ferrocyanide, 25 mM potassiumferricyanide, 100 mM CAPS, pH 10.0) withalpha-hemolysin-(E111N/K147N)₇(SEQ ID NO: 5 and 6) was flowed over thearray. A holding potential of +180 mV was applied and pores were allowedto enter bilayers until at least 10% occupancy was achieved. Once poreshad inserted, then buffer solution (625 mM KCl, 75 mM potassiumferrocyanide, 25 mM potassium ferricyanide, 100 mM CAPS, pH 10.0)containing no alpha-hemolysin-(E111N/K147N)₇(SEQ ID NO: 5 and 6) wasthen flowed over the array to prevent further pores inserting into thetri-block co-polymer. In order to observe thrombin binding to anaptamer, a solution containing the aptamer (SEQ ID NO: 7, 1 μM) andthrombin (1 μM) in buffer (625 mM KCl, 75 mM potassium ferrocyanide, 25mM potassium ferricyanide, 100 mM CAPS, pH 10.0) was flowed over thearray. A holding potential of +180 mV was applied and characteristicblock levels corresponding to the presence and absence of thrombin weredetected.

Results

Upon the exposure of the tri-block co-polymer covered agarose dropletsto alpha-hemolysin-(E111N/K147N)₇(SEQ ID NO: 5 and 6) nanopores,insertion of the pores into the tri-block co-polymer was observed. Onthe addition of thrombin and aptamer (SEQ ID NO: 7) to the system,characteristic block levels corresponding to the presence and absence ofthrombin were observed. Current traces were obtained showing the blockproduced in the absence of bound thrombin (1) and in the presence ofthrombin (2), as shown in FIG. 49 which is a current trace (which islow-pass filtered) showing characteristic block levels corresponding tothe presence (block labeled2) and absence (block labeled1).

EXAMPLE 7

This example describes how optical measurements were used to determinewhether MspA-(B2C) (SEQ ID NO: 1 and 9) pores had inserted into triblockcopolymer droplets.

Materials and Methods 7.1—Droplet Formation

With the ExoI/DNA buffer 1 (962.5 μM KCl, 7.5 mM potassium ferrocyanide,2.5 mM potassium ferricyanide, 100 mM CAPS (pH10), 50 μM EDTA, 50 nM EcoExoI, 5 μM FAM/BHQ1-labeled PolyT 30 mer (SEQ ID NO: 8)) and triblockcopolymer (6-30-6) in AR20 oil in separate 1 mL Hamilton syringes,droplets were prepared by flowing at 16 μL/min (buffer 1) and 4 4/min(triblock copolymer in oil), respectively through a Dolomite T-piece.

7.2—Array Population and Pore Insertion

Using a 200 μL pipette tip with the end cut off, 200 μL of droplets werepipette onto four clean arrays. Excess droplets were washed off with 2mg/mL Triblock 6-30-6 in oil. 500 μL of buffer 2 (962.5 μM KCl, 7.5 mMpotassium ferrocyanide, 2.5 mM potassium ferricyanide, 100 mM CAPS(pH10), 50 μM EDTA) was then flowed over each of the four arrays inorder to cover the droplets. Brightfield images of each array wereobtained using the fluorescence microscope.

Buffers 3 and 4 were then prepared as shown in the Table 2 below. Buffer3 (which contained MspA-(B2C) nanopores) (500 μL) was flowed over twoarrays and Buffer 4 (which contained no nanopores as a control) wasflowed over the other two arrays. Buffer 3 and 4 were left on the arraysfor 30 minutes before Mg2+ containing buffer (buffer 5—0.5 M MgCl₂, 100mM CAPS, pH10, 7.5 mM potassium ferrocyanide, 2.5 mM potassiumferricyanide) was flowed across all four arrays. The arrays were thenleft overnight at room temperature before acquiring Brightfield and FITC(2 s exposure) images of each array using a 5× lens.

TABLE 2 Buffer 3 Buffer 4 MspA-(B2C)   7.5 μL Storage buffer —   7.5 μLBuffer 2 1492.5 μL 1492.5 μL Storage buffer = 50 mM Tris HCl, pH 9.0,100 mM NaCl, 0.1% DDM

Results

This example describes how optical measurements can be used to determinewhether MspA-(B2C) (SEQ ID NO: 1 and 9) pores have inserted intotriblock copolymer droplets. MspA-(B2C) (SEQ ID NO: 1 and 9) pores wereallowed to insert into triblock copolymer droplets, which contained ExoIenzyme and fluorphore/quencher-labeled DNA substrate (SEQ ID NO: 8). Bysubsequently flowing a Mg²⁺-containing buffer across the top of thedroplets, flow of Mg²⁺ cations into the droplets, through insertednanopores, activated the ExoI, allowing it to digest thefluorophore/quencher DNA, resulting in a fluorescence increase. Thearrays that were treated with MspA-(B2C) (SEQ ID NO: 1 and 9) containingbuffer (buffer 3) showed bright spots on the arrays which indicates thatpores have inserted into the droplets, as shown in FIG. 50. FIG. 51shows a control experiment where buffer which contained no MspA-(B2C)(SEQ ID NO: 1 and 9) (buffer 4) was used. The absence of bright spotsshows that under control conditions (absence of MspA-(B2C) nanopores)Mg²⁺ cannot penetrate the triblock copolymer, therefore, preventingactivation of the enzyme and an increase in fluorescence. By comparisonof FIG. 50 and FIG. 51 it is clear that the droplets which were exposedto buffer containing nanopores showed bright spots which corresponded toinsertion of nanopores into the triblock copolymer.

EXAMPLE 8

This example describes the method used to populate the arrays, whichwere assembled with patterned interconnecting droplet zones.

Materials and Methods 8.2 Membrane Formation on Semi-Closed StructureArrays

Using a micropipette, 504 of a 1504 AR20/1 ml hexane mixture wasdispensed onto the surface of a dry array at a temperature of 100° C.and left for 1 h to allow the oil to be distributed through the arraysurface by capillarity and for the hexane to evaporate. The array wasmounted on an array holder and a 1.5 mm thick gasket was placed on it,aligned in such a way that the array was completely open and surrounded.The buffer intended to fill in each of the individual wells was thendispensed on top of the array (700 μL); the gasket should contain thebuffer volume. The array was then placed in a vacuum chamber and pumpeddown to 25 mbar for 1 min such that volumes of buffer were provided inthe wells It was then removed from the vacuum chamber and placed on aflow cell assembly clamp, where a flow cell was aligned to the holderand clamped to seal the assembly. An AR20 flow-front (700 μL) was thenslowly pushed through the flow cell with a pipette; in this step theindividual aqueous volumes contained within the wells were separatedfrom the bulk and encapsulated in oil. This step was followed by a 5 mLair flow-front which displaced the excess oil out of the flow cell. Theflow cell was then unclamped and disassembled allowing 30 μL of oil witha 10 mg/mL concentration of tri-block co-polymer (TBCP) to be dispensedon top of the array and left to incubate for 20 min. After theincubation step the excess oil was removed by placing the array at 90°and allowing it to flow off the array so it can be dried with a tissue.At this stage the aqueous volumes were ready to form TBCP membranes.

Once the wells had been filled with aqueous and TBCP had been introducedinto the system the array was then assembled into an assay flow cell,where buffer was then introduced. As the buffer flow front travelledover the array, it displaced any excess oil left allowing the bulkbuffer volume to form TBCP membranes.

EXAMPLE 9

This example describes the method used to populate arrays with volumesof polar and apolar media according to that shown in FIGS. 21 and 22 andas shown schematically FIG. 32.

Oil Pretreatment

An array was subjected to an oil preconditioning with a small amount ofAR-20 silicone oil to fill the micro-patterning of the well and coverthe pillars and surface in a thin oil film. A 1 mL syringe barrel of aHarvard syringe pump was primed with AR-20 oil and the dispense speedset to 2 μl/sec. 1.7 μl of AR20 silicone oil was dispensed onto thecentre of a hexagonal close packed array of dimensions 6.04 mm×14.47 mmhaving 2048 compartments spaced with a pitch of 200 μm, a well height of90 μm and a pillar height of 30 μm and allowed to spread through thearray. The array was then subjected to 100 deg. C in an oven for 30 minsand subsequently removed and allowed to cool. The array was inspected toensure the oil had reached the edges of the array before use.

Buffer Filling

10 ml of buffer (600 mM KCl, 100 mM Hepes, 75 mM Potassium Ferrocyanide(II), 25 mM Potassium Ferricyanide (III), pH 8) was degassed and loadedinto a flow-cell reservoir. The array was placed in the flow-cell asshown in FIG. 27 and the array was filled with buffer under vacuum(approx. 35 mBar) to provide volumes of buffer in the compartments.

Oil Filling

Immediately following the buffer filling step, 5 μl of 10 mg/mlTBCP/AR-20 was added to the flow-cell and flowed over the top of thearray under vacuum. This was left to incubate for approx. 5 minute toensure that the TBCP covered the entire array. Excess buffer was removedfrom the non-array areas and excess oil was removed from the array undervacuum.

Addition of Buffer Layer

Following the oil filling step, a further amount of buffer was flowedover the array in order to provide a buffer layer/TBCP/volume of bufferinterface. The layer of buffer also minimises evaporation of water fromthe volumes of buffer in the compartments.

EXAMPLE 10

This example describes how the method used to populate the arraysdescribed in Example 8 was modified in order to produce confocalmicroscopy images showing the uniform population of the interconnectingdroplet zones and membrane formation. The images show that the aqueousvolumes pinned to the walls of the wells resulting in the control ofmembrane size.

Materials and Methods

The various images described above were taken by confocal imaging. Inorder to render the materials involved in the experimentsdistinguishable in confocal microscopy, fluorescent dyes were diluted inthe reagents. The oil (AR20) was dyed with BODIPY 493/503 (green) andthe buffer solution, which formed the discrete volumes, was dyed withSulforhodamine B (red). The remaining materials were not dyed andtherefore appear as dark regions in the confocal images. In membraneformation experiments (shown in FIGS. 39 and 40) the incubation oil,with 10 mg/mL of TBCP, was also dyed with BODIPY 493/503. The bulkbuffer which was flowed over the array after the first aqueous volumehad pinned to the walls of the inner wells was not dyed. The confocalmicroscopy samples were prepared with the above reagents using themethod described in the previous section (Example 8), and then imagedusing a Nikon Al Confocal Microscope.

1. A method of forming an array of membranes comprising amphipathicmolecules, the method comprising: providing an apparatus comprising asupport defining an array of compartments having openings through whichpolar medium may be introduced; disposing polar medium and apolar mediumonto the support to provide volumes comprising polar medium withinrespective compartments so that the volumes comprising polar medium areconstrained from contacting volumes comprising polar medium inneighbouring compartments, and a layer comprising apolar mediumextending across the openings in the support in contact with the volumescomprising polar medium; and flowing polar medium across the openings inthe support to displace apolar medium and form a layer comprising polarmedium extending across the openings in the support in contact with thevolumes comprising polar medium and membranes comprising amphipathicmolecules at the interfaces between the layer comprising polar mediumand the volumes comprising polar medium.
 2. A method according to claim1, wherein the step of disposing polar medium and apolar medium onto thesupport comprises: disposing polar medium onto the support so that thepolar medium enters into the compartments through the openings; andsubsequently providing said layer comprising apolar medium.
 3. A methodaccording to claim 2, wherein the step of disposing polar medium ontothe support comprises flowing polar medium across the support so thatthe polar medium enters into the compartments through the openings.
 4. Amethod according to claim 3, wherein the step of disposing polar mediumand apolar medium onto the support comprises, between said step offlowing polar medium across the support and said step of providing saidlayer comprising apolar medium, flowing a gas across the support todisplace excess polar medium, leaving the volumes comprising polarmedium in the compartments.
 5. A method according to claim 3, whereinthe step of providing said layer comprising apolar medium comprisesflowing apolar medium across the support to displace excess polar mediumleaving the volumes comprising polar medium in the compartments and thelayer comprising apolar medium extending across the openings in thesupport in contact with the volumes comprising polar medium.
 6. A methodaccording to claim 1, wherein the step of disposing polar medium andapolar medium onto the support comprises injecting volumes of polarmedium into the respective compartments.
 7. A method according to claim6, wherein the step of disposing polar medium and apolar medium onto thesupport further comprises, subsequently to injecting volumes of polarmedium into each compartment, providing said layer comprising apolarmedium.
 8. A method according to any one of claims 2 to 7, wherein saidstep of providing said layer comprising apolar medium comprises flowingapolar medium across the support.
 9. A method according to any one ofclaims 2 to 8, further comprising pre-treating the support with apre-treatment of apolar medium before said step of disposing polarmedium and apolar medium onto the support.
 10. A method according toclaim 9 wherein the pre-treatment of apolar medium does not compriseamphipathic molecules.
 11. A method according to any one of claims 2 to10, wherein the layer comprising apolar medium further comprises theamphipathic molecules.
 12. A method according to any one of claims 2 to10, wherein the polar medium that is flowed across the openings in thesupport further comprises the amphipathic molecules.
 13. A methodaccording to claim 1, wherein said step of disposing polar medium andapolar medium onto the support comprises forming an emulsion of volumescomprising polar medium in an apolar medium and flowing the emulsionover the support to provide the volumes comprising polar medium withinthe apolar medium in the compartments through the openings.
 14. A methodaccording to claim 13, wherein the emulsion further comprises theamphipathic molecules.
 15. A method according to claim 13 or 14, whereinthe emulsion contains a number of volumes comprising polar medium whichis in excess of the number of compartments.
 16. A method according toclaim 15, wherein said step of disposing polar medium and apolar mediumonto the support further comprises washing the support with the apolarmedium to remove the excess volumes comprising polar medium, leaving alayer of the apolar medium used for washing as said layer of apolarmedium extending across the openings in the support in contact with thevolumes comprising polar medium.
 17. A method according to any one ofthe preceding claims, wherein the support comprises a base andpartitions extending from the base that define the compartments andconstrain the volumes comprising polar medium from contacting volumescomprising polar medium in neighbouring compartments.
 18. A methodaccording to claim 17, wherein the partitions have gaps that allow theflow of apolar medium between the compartments.
 19. A method accordingto claim 18, wherein the partitions comprise inner portions and outerportions, the inner portions defining inner recesses of the compartmentswithout gaps therebetween, the volumes comprising polar medium beingdisposed within the inner recesses of the respective compartments, andthe outer portions extending outwardly from the inner portions definingouter portions of the compartments and in which the gaps that allow theflow of apolar medium between the compartments are formed.
 20. A methodaccording to claim 19, wherein the volumes comprising polar medium fillthe inner recesses, and the inner recesses and the outer portions of thepartitions have dimensions selected so that the volumes comprising polarmedium form a meniscus across the inner recess and the layer comprisingpolar medium forms meniscuses across the outer portions, whichmeniscuses extend towards each other to an extent that brings the layercomprising polar medium in contact with the volumes comprising polarmedium.
 21. A method according to claim 19 or 20, wherein the outerportions are pillars extending from the inner portion.
 22. A methodaccording to any one of claims 19 to 21, wherein the outer portions areset back from the edges of the inner recesses as viewed from theopenings.
 23. A method according to any one of claims 19 to 22, whereinthe inner recesses have surfaces having a patterning that is arranged toretain apolar medium.
 24. A method according to claim 23, wherein thepatterning comprises a plurality of indentations that extend outwardlyof the inner recesses.
 25. A method according to any one of claims 19 to24, wherein the outer portions comprise have surfaces having apatterning that is arranged to retain apolar medium.
 26. A methodaccording to claim 25, wherein the patterning comprises a plurality ofindentations that extend outwardly of the inner portions of thecompartments.
 27. A method according to any one of claims 19 to 26,wherein the inner portions of the partitions are formed by a common bodyextending from the base.
 28. A method according to any one of claims 19to 27, wherein the base has planar surfaces forming the inner ends ofthe compartments.
 29. A method according to any one of claims 19 to 28,wherein the inner recesses have a profile as viewed from the openingsthat is circular around individual compartments.
 30. A method accordingto claim 19, wherein the gaps that allow the flow of apolar mediumbetween the compartments extend to the base.
 31. A method according toclaim 17, wherein the partitions have no gaps allowing the flow ofapolar medium between the compartments.
 32. A method according to anyone of claims 17 to 31, wherein the outer ends of the partitions extendin a common plane.
 33. A method according to any one of the precedingclaims, wherein the layer comprising polar medium comprises a sample tobe analysed.
 34. A method according to any one of claims 1 to 32,further comprising displacing the layer comprising polar medium byfurther polar medium comprising a sample to be analysed.
 35. A methodaccording to any one of the preceding claims, further comprising causingmembrane proteins to insert into the membrane comprising amphipathicmolecules.
 36. A method according to claim 35, wherein the membraneproteins are ion channels or pores.
 37. A method according to claim 35or 36, wherein the layer comprising polar medium contains the membraneproteins which are capable of insertion into a layer of said amphipathicmolecules.
 38. A method according to any one of the preceding claims,further comprising taking electrical measurements between the layercomprising polar medium and the respective volumes comprising polarmedium
 39. A method according to claim 38, wherein the electricalmeasurements are dependent on a process occurring at or through themembranes comprising amphipathic molecules.
 40. A method according toany one of the preceding claims, wherein the support further comprisesrespective electrodes in each compartment making electrical contact withthe volumes comprising polar medium, the step of disposing disposingpolar medium and apolar medium onto the support provides volumescomprising polar medium within respective compartments in electricalcontact with the respective electrodes.
 41. A method according to anyone of the preceding claims, wherein the apolar medium comprises an oil.42. A method according to any one of the preceding claims, wherein thevolumes comprising polar medium are volumes comprising an aqueousmedium.
 43. A method according to any one of the preceding claims,wherein the volumes comprising polar medium have an average volume inthe range from 0.4 pL to 400 nL.
 44. A method according to any one ofthe preceding claims, wherein the respective compartments are providedwith a single volume comprising polar medium.
 45. An apparatus forforming an array of volumes comprising polar medium, the apparatuscomprising a support that comprises partitions which comprise innerportions and outer portions, the inner portions defining inner recesseswithout gaps therebetween that are capable of constraining volumescomprising polar medium that may be contained in neighbouring innerrecesses from contacting each other, and the outer portions extendingoutwardly from the inner portions and having gaps allowing the flow ofan apolar medium across the substrate.
 46. An apparatus according toclaim 45, wherein the outer portions are pillars extending from theinner portion.
 47. An apparatus according to claim 45 or 46, wherein theouter portions are set back from the edges of the inner recesses asviewed towards the openings
 48. An apparatus according to any one ofclaims 45 to 47, wherein the inner recesses have surfaces having apatterning that is arranged to retain apolar medium.
 49. A methodaccording to claim 48, wherein the patterning comprises a plurality ofindentations that extend outwardly of the inner recesses.
 50. Anapparatus according to any one of claims 45 to 49, wherein the outerportions have surfaces having a patterning that is arranged to retainapolar medium.
 51. A method according to claim 50, wherein thepatterning comprises a plurality of indentations that extend outwardlyof the inner portions of the compartments.
 52. An apparatus according toany one of claims 45 to 51, wherein the inner portions of the partitionsare formed by a common body.
 53. An apparatus according to any one ofclaims 45 to 52, wherein the inner ends of the recesses are planarsurfaces.
 54. An apparatus according to any one of claims 45 to 53,wherein the inner portions of the partitions have a non-circular profileas viewed from the openings that comprises, around individualcompartments, one or more salient portions for constraining a volumecomprising polar medium and one or more re-entrant portions providingchannels allowing outflow of apolar medium displaced by entry of avolume comprising polar medium into the compartment.
 55. An apparatusaccording to any one of claims 45 to 54, wherein the inner portions ofthe partitions have a profile as viewed from the openings that iscircular around individual compartments.
 56. An apparatus according toany one of claims 45 to 55, wherein the outer ends of the partitionsextend in a common plane.
 57. An apparatus according to claim 56,wherein the total length per compartment of the edges of the outer endsof the partitions in the common plane is greater than the largestcircumference of the largest notional sphere that can be accommodatedwithin the compartments.
 58. An apparatus according to any one of claims45 to 57, wherein the inner recesses are capable of containing volumescomprising polar medium that have an average volume in the range from0.4 pL to 400 nL.
 59. An apparatus according to any one of claims 45 to58, wherein the inner recesses contain respective volumes of polarmedium.
 60. An apparatus according to claim 59, further comprising alayer comprising apolar medium extending across the support in contactwith the volumes comprising polar medium.
 61. An apparatus according toclaim 59, further comprising a layer comprising polar medium extendingacross the support with apolar medium around the outer portions of thepartitions and with the layer of polar medium in contact with thevolumes comprising polar medium, membranes comprising amphipathicmolecules being formed at the interfaces between the layer comprisingpolar medium and the volumes comprising polar medium.
 62. An apparatusaccording to any one of claims 45 to 61, wherein the inner recesses arecapable of containing a single volume comprising polar medium.